Abstract

Accumulation of reactive oxygen species is a common phenomenon in cardiac stress conditions, for instance, coronary artery disease, aging-related cardiovascular abnormalities, and exposure to cardiac stressors such as hydrogen peroxide (H2O2). Mitochondrial protein 18 (Mtp18) is a novel mitochondrial inner membrane protein, shown to involve in the regulation of mitochondrial dynamics. Although Mtp18 is abundant in cardiac muscles, its role in cardiac apoptosis remains elusive. The present study aimed to detect the role of Mtp18 in H2O2-induced mitochondrial fission and apoptosis in cardiomyocytes. We studied the effect of Mtp18 in cardiomyocytes by modulating its expression with lentiviral construct of Mtp18-shRNA and Mtp18 c-DNA, respectively. We then analyzed mitochondrial morphological dynamics with MitoTracker Red staining; apoptosis with terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) and cell death detection assays; and protein expression with immunoblotting. Here, we observed that Mtp18 could regulate oxidative stress- mediated mitochondrial fission and apoptosis in cardiac myocytes. Mechanistically, we found that Mtp8 induced mitochondrial fission and apoptosis by enhancing dynamin-related protein 1 (Drp1) accumulation. Conversely, knockdown of Mtp18 interfered with Drp1-associated mitochondrial fission and subsequent activation of apoptosis in both HL-1 cells and primary cardiomyocytes. However, overexpression of Mtp18 alone was not sufficient to execute apoptosis when Drp1 was minimally expressed, suggesting that Mtp18 and Drp1 are interdependent in apoptotic cascade. Together, these data highlight the role of Mtp18 in cardiac apoptosis and provide a novel therapeutic insight to minimize cardiomyocyte loss via targetting mitochondrial dynamics.

Introduction

Apoptosis plays a pivotal role in both cardiac physiology and pathology. Programmed apoptosis is essential in shaping the heart and vasculatures during early morphogenesis [1], on the other hand, overactivation of apoptosis is related to various cardiovascular pathologies including myocardial infarction, cardiomyopathy, and cardiac hypertrophy [2–4]. Being the highly energy demanding organ, the heart is abundant in mitochondria [5]. Mitochondria are active regulators of apoptosis. Several apoptotic signals converge at mitochondria and their interactions at the mitochondrion ultimately determine whether a cell will survive or die in response to various physiologic or therapeutic cell death stimuli [6–8]. During the event of executing the death sentence, mitochondria undergo dramatic morphological changes comprising mitochondrial fragmentation and cristae remodeling [6,9]. The fragmentation of mitochondria is essential for increasing the mitochondrial outer membrane permeability, which subsequently causes the release of mitochondrial pro-apoptotic factors such as cytochrome c (Cyt-c) into the cytosol and further activates the downstream apoptotic cascades [10,11]. In addition, studies indicated that inhibition of mitochondrial fission can protect the heart from apoptosis during ischemia/reperfusion injury [12,13]. Yet, the relationship between mitochondrial dynamics and apoptosis control has not been fully verified.

Mitochondrial membrane proteins such as dynamin-related protein 1 (Drp1) and fission protein 1 (Fis1) are the principal mediators of mitochondrial fission [14,15]. Knockdown of Drp1 prevents mitochondrial fission and inhibits apoptosis activation [16,17]. Contrariwise, overexpression of Fis1 induces mitochondrial fission, resulting in mitochondrial fragmentation and subsequent apoptosis. Although multiple fusion and fission proteins have been identified, their molecular mechanisms are still under exploration [11]. A novel mitochondrial inner membrane protein, known as mitochondrial protein 18 (Mtp18), was shown to involve in the regulation of mitochondrial dynamics in cancer cells [18,19]. Mtp18 is abundant in cardiac and skeletal muscles. Our previous work indicated that Mtp18 expression was up-regulated in cardiac myocytes during doxorubicin (Dox) treatment and responsible for mitochondrial fission and apoptosis [20]. In contrast, in cancer cells, we found that Dox down-regulated Mtp18 expression [21]. These findings suggest that the molecular regulation of Mtp18 is cell-type specific.

Reactive oxygen species (ROS) accumulation can trigger apoptosis in cardiomyocytes [22,23]. ROS can be formed in the heart as a byproduct of aerobic metabolism or can also be induced by inflammatory markers such as cytokines and growth factors, etc. ROS formation is generally counterbalanced by several antioxidants and redox regulatory system [24]. However, long-term ROS accumulation in the cardiovascular system is seen in cardiac stress conditions including coronary artery disease [25], aging-related cardiovascular abnormalities [24,26], and exposure to cardiac stressors such as hydrogen peroxide (H2O2) and doxorubicin [23,27]. ROS accumulation induces mitochondrial fission and further activates the mitochondrial pathway of apoptosis [22–24]; the molecular mechanisms underlying oxidative stress-induced cardiomyocyte apoptosis have not yet been fully elucidated. In order to understand the molecular mechanism of cardiac apoptosis, here, we aimed to detect the role of mitochondrial fission in H2O2-induced apoptosis in cardiomyocytes focusing on Mtp18.

Materials and methods

Cell cultures and hydrogen peroxide treatment

Primary rat cardiac myocytes (Lonza, Walkersville, MD, U.S.A.) were cultured in Rat Cardiac Myocyte Growth Media (RCGM; Lonza, Walkersville, MD, U.S.A.) containing horse serum, fetal bovine serum (FBS), and gentamicin/amphotericin-B, further supplemented with 200 µM 5- bromo- 2’-deoxyuridine (BrdU) in a humidified 5% CO2 incubator at 37°C. HL-1 is a cardiac muscle cell line which derived from the AT-1 mouse atrial cardiomyocyte tumor lineage. HL-1 cell lines, derived from the AT-1 mouse atrial cardiomyocyte tumor lineage, were proved to retain the phenotypic characteristics of cardiomyocytes after serially passaged [26]. The cells were cultured in Claycomb media supplemented with 10% FBS (Sigma–Aldrich, St. Louis, MO, U.S.A.), 0.1 mol/l of norepinephrine (Sigma–Aldrich, St. Louis, MO, U.S.A.), 2 mmol/l of L-glutamine (Invitrogen, Carlsbad, CA, U.S.A.), and penicillin/streptomycin ((Invitrogen, Carlsbad, CA, U.S.A.) in a humidified 5% CO2 incubator at 37°C [28]. H2O2 treatment was performed as described elsewhere [29]. In brief, when the cultured cells reached approximately 60% confluently, they were incubated at 37°C for 1–24 h in complete culture medium containing the indicated concentrations of H2O2. The concentration of hydrogen peroxide varied from 50–200 µM depending on the purpose and duration of apoptosis induction.

The lentiviral construct of Mtp18-shRNA and infection

Mtp18-shRNA and control shRNA lentivirus-based plasmid were purchased Origene (Rockville, MD, U.S.A.). The detailed procedure of transfection is as described in our previous work [20]. Briefly, viruses were amplified in 293T cells. Cells were infected with the virus according to manufacturer’s protocol. Mtp18-shRNA contains four specific constructs, encoding shRNA designed to knockdown Mtp18 expression and control shRNA lentiviral-based plasmid includes a shRNA construct encoding a scrambled sequence that will not lead to specific degradation of any known cellular mRNA. Among four different shRNA-plasmids, Mtp18-shRNA-D showed the most significant knockdown effect on Mtp18 expression. Therefore, we chose to use Mtp18-shRNA-D in the subsequent experiments. The Mtp18- shRNA-D and scramble shRNA sequences were similar to as we have described [20]. We infected the cells with the virus according to manufacturer’s protocol. The Mtp18 expression levels were determined with western blot.

The constructs of Mtp18 and Drp1 and infection

We purchased lentiviral particle harboring the cDNA of Mtp18 (RR203770L2) and cDNA of Drp1 (MR210122L2) from Origene (Rockville, MD, U.S.A.). We amplified viruses in 293T cells. We infected the cells with the virus according to manufactural protocol. The Mtp18 and Drp1 expression levels were determined using western blot.

Drp1 siRNA Transfection

We purchased the Drp1 siRNA and control siRNA-A (scrambled siRNA) from Santa Cruz Biotechnology, Inc. (Dallas, TX, U.S.A.). Drp1 siRNA is a pool of three target-specific 19-25 nt siRNAs designed to knock down Drp1 gene expression. The sequences of Drp1 siRNA-A, -B, and -C and those of the scrambled siRNA as well as the transfection procedure are similar to as we have described [20]. Briefly, cells were seeded 24 h before transfection and then transfected with 30 nM scrambled siRNA or Drp1 siRNA using Lipofectamine® 2000 Reagent (Invitrogen, Carlsbad, CA, U.S.A.) per the manufacturer’s instructions.

Immunoblotting

The immunoblotting procedure is similar to as we have reported earlier [29]. In brief, we lysed the cells for 30 min on ice in RIPA buffer (Invitrogen, Carlsbad, CA, U.S.A.) and a protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO, U.S.A.). We separated the samples using SDS–PAGE using a 4–20% Mini-PROTEAN TGX™ Precast Gel (Bio-Rad, Hercules, CA, U.S.A.) and transferred for 1 h at 80V to PVDF membrane (Merck Millipore Ltd, Darmstadt, Germany). Equal protein loading was determined by Ponceau red staining. We blocked the membrane in Tris-buffered saline containing 5% milk and 0.1% of Tween 20 for 1 h and then incubated overnight with primary antibody. We probed the blots with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG. (Santa Cruz Biotechnology, Inc., Dallas, TX, U.S.A.). We visualized antigen-antibody complexes using AmershamTM ECLTM Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, U.K.). Anti-Mtp18, anti-caspase-3 polyclonal, and anti-cleaved poly ADP ribose polymerase (PARP) monoclonal antibodies were from Abcam (Cambridge, MA, U.S.A.). Anti-actin monoclonal and Anti-DRP1 polyclonal antibodies were from Santa Cruz Biotechnology, Inc. Anti- tubulin polyclonal, anti-cyclooxygenase IV and anti-cytochrome c monoclonal, and anti-phospho-Drp1 (Ser637) polyclonal antibodies were from Cell Signaling Technology, Inc. (Danvers, MA, U.S.A.). The protein band intensity was quantitated by ImageJ (National Institutes of Health, Bethesda, Maryland, U.S.A.) using protocol written by Luke Miller. Briefly, we quantitated the density of each sample with ImageJ, then the percent value of each sample and that of the standard was calculated. Finally, the relative density was calculated by dividing the percent value of each sample by the percent value of each standard [29].

DNA fragmentation and apoptosis assays

DNA fragmentation was monitored using the cell death detection ELISA kit (Roche, Branford, CT, U.S.A.) as we previously described [30]. Briefly, we added the anti-histone monoclonal antibody to the 96-well ELISA plates and incubated the plates overnight at 4°C. After recoating and rinsing three times, we added the cytoplasmic fractions and incubated for 90 min at room temperature (RT). After three washes, we detected the bound nucleosomes by the addition of anti-DNA peroxidase monoclonal antibody and reacted for 90 min at RT. After the addition of the substrate, the optical density was determined at 405 nm using an ELISA plate reader. For apoptosis analysis, we used a terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) kit from Abcam (Cambridge, MA, U.S.A.). After transfection and treatment, we rinsed, fixed, permeabilized, and stained the cells with the in situ BrdU-red DNA fragmentation TUNEL assay according to the kit’s instructions. We took the images using a laser scanning confocal microscope (Zeiss LSM 710 BIG, Dublin, CA, U.S.A.). For each group, we counted approximately 250–300 cells in 20–30 random fields. Results are expressed as percentages of TUNEL positive cells.

Preparation of mitochondrial fractions

We prepared mitochondrial fractions as previously described [30]. Briefly, we washed the cells twice with PBS and suspended the pellet in 0.2 ml of buffer A (20 mM HEPES pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, and 250 mM sucrose) containing a protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO, U.S.A.). We homogenized the cells by 12 strokes in a Dounce homogenizer. We centrifuged the homogenates twice at 750 g for 5 min at 4°C to collect nuclei and debris; we centrifuged the supernatant at 10000 g for 15 min at 4°C to obtain mitochondria-enriched heavy membranes (HM). We centrifuged the resulting supernatants to yield cytosolic fractions.

Analysis of mitochondrial fission

We analyzed mitochondrial fission by staining mitochondria as we have described earlier with some modification [31]. Briefly, we plated the cells onto the coverslips and exposed to the respective treatment. Then, we stained the coverslips with 100 M MitoTracker Red CMXRos (Molecular Probes, Eugene, OR, U.S.A.) for 15 min and fixed in 4% paraformaldehyde for 15 min. Then we permeabilized the coverslips with 0.2% Triton X-100. We imaged mitochondria using a laser scanning confocal microscope (Zeiss LSM 710 BIG, Dublin, U.S.A.). The detailed procedure of analysis of mitochondrial morphology was as described [31]. Mitochondrial fission is identified by cells with disintegrated mitochondria. The percentage of cells with fragmented mitochondria relative to the total number of cells is presented as the mean ± S.E.M. of at least three independent experiments, counted by an observer blinded to the experimental conditions. We counted 200–300 cells in 20–30 random fields per group.

Electron Microscopy

We prepared samples and carried out conventional electron microscopy (EM) as described [31]. We examined samples at a magnification of ×15,000 with a JEOL JEM-1230 transmission electron microscope. For comparison of mitochondrial fission, we evaluated EM micrographs of thin sections. We measured the sizes of individual mitochondria using Image Pro Plus software. For each experiment, we measured approximately 100 mitochondria in a representative area to determine the percentages of mitochondria with various sizes. We categorized mitochondria smaller than 0.6 μm2 as having undergone fission.

Prediction of a potential Mtp18’s target protein

The potential target protein was predicted using STRING v10 (http://string-db.org/cgi/input.pl). The search term was set as ‘Mtp18’ and organism as ‘Mus musculus.’ The protein–protein interaction was determined by the interaction score, which is an indicator of confidence regarding how likely STRING judges an interaction to be true, given the available evidence [32]. The score can range from 0 to 1, with 1 being the highest possible confidence [33].

Statistical analysis

Data are expressed as the mean ± S.E.M. of at least three independent experiments for each experimental group. We evaluated the data with Student’s t test for comparisons between two groups and one-way analysis of variance for multiple comparisons. Statistical analyses were performed with GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA). A value of P<0.05 was considered significant.

Results

H2O2 exposure induces apoptosis in cardiac myocytes

To understand whether Mtp18 is involved in the regulation of oxidative stress-induced cardiomyocyte mitochondrial fission and activation of apoptotic signaling, we treated the HL-1 cells with H2O2 and determined the extent of apoptosis. We detected the level of cleavage of caspase-3 and PARP at different time points of H2O2 treatment. We observed a time-dependent increase in the cleavage of caspase-3 and PARP upon H2O2 exposure (Figure 1A).

Hydrogen peroxide exposure induces apoptosis in cardiac myocytes

Figure 1
Hydrogen peroxide exposure induces apoptosis in cardiac myocytes

(A) Shows caspase-3 and PARP1 cleavage by immunoblot (left panel) and densitometry (right panel). β-actin served as a loading control. HL-1 cells were stimulated with H2O2 and then harvested at the indicated time for immunoblotting. The densitometry data were expressed as the mean ± S.E.M. of three independent experiments. (B) Shows the release of cytochrome-c (Cyt-c) from mitochondria into cytosol upon H2O2 treatment by immunoblotting (up panel) and densitometry (low panel). Number on the right indicates the molecular weight (kDa). β-actin served as a loading control for whole cell lysate. Cytochrome c oxidase (Cox4) served as a loading control for HM (mitochondrial enriched heavy membrane fraction). Cells were stimulated with H2O2 and then harvested at the indicated time for subcellular fraction and immunoblotting. The densitometry data were expressed as the mean ± S.E.M. of three independent experiments. (C,D) Show DNA fragmentation. Cells were stimulated with the indicated dose of H2O2 for 24 h (C) or with 200 µmol/l H2O2 at the indicated time points (D), and apoptosis-related DNA fragmentations were analyzed using the cell death detection ELISA. Data were expressed as the mean ± S.E.M. of three independent experiments. Figures presented are the representative figures of at least three independent experiments. **P<0.01, ***P<0.001, and ****P<0.0001 vs 0 h.

Figure 1
Hydrogen peroxide exposure induces apoptosis in cardiac myocytes

(A) Shows caspase-3 and PARP1 cleavage by immunoblot (left panel) and densitometry (right panel). β-actin served as a loading control. HL-1 cells were stimulated with H2O2 and then harvested at the indicated time for immunoblotting. The densitometry data were expressed as the mean ± S.E.M. of three independent experiments. (B) Shows the release of cytochrome-c (Cyt-c) from mitochondria into cytosol upon H2O2 treatment by immunoblotting (up panel) and densitometry (low panel). Number on the right indicates the molecular weight (kDa). β-actin served as a loading control for whole cell lysate. Cytochrome c oxidase (Cox4) served as a loading control for HM (mitochondrial enriched heavy membrane fraction). Cells were stimulated with H2O2 and then harvested at the indicated time for subcellular fraction and immunoblotting. The densitometry data were expressed as the mean ± S.E.M. of three independent experiments. (C,D) Show DNA fragmentation. Cells were stimulated with the indicated dose of H2O2 for 24 h (C) or with 200 µmol/l H2O2 at the indicated time points (D), and apoptosis-related DNA fragmentations were analyzed using the cell death detection ELISA. Data were expressed as the mean ± S.E.M. of three independent experiments. Figures presented are the representative figures of at least three independent experiments. **P<0.01, ***P<0.001, and ****P<0.0001 vs 0 h.

It is well established that activation of the mitochondrial pathway of apoptosis eventually leads to the release of cytochrome-c (Cyt-c) from mitochondrial intermembrane space into the cytosol. Therefore, we performed the subcellular fraction and analyzed the Cyt-c distribution between mitochondrial and cytosolic fraction. H2O2 exposure induces a time-dependent increase in Cyt-c expression and increase in the release of Cyt-c into the cytosol (Figure 1B). Concurrently, we observed a dose- and time-dependent increase in apoptosis-related DNA fragmentation in the cell death detection ELISA assay (Figure 1C,D) and a concomitant decrease in cell viability (Supplementary Figure S1A,B).

H2O2-induced mitochondrial fission is associated with up-regulation in Mtp18 expression

To understand the role of Mtp18 in oxidative stress induced mitochondrial fission, we examined the mitochondrial morphology upon H2O2 exposure. As shown in Figure 2A, when compared with negative control (where the mitochondria are long, thin, and filamentous), the H2O2-treated group displayed punctate disintegrated mitochondria, undergoing fission. In quantitative analysis, a time-dependent increase in the percentages of cells with mitochondrial fission upon H2O2 exposure was observed (Figure 2B). These findings confirmed that H2O2 induces mitochondrial fission and apoptosis in HL-1 cells. Recent studies have reported that Mtp18 plays a critical role in the regulation of mitochondrial fission in various cancer cells [18,19]. However, its role in the cardiomyocyte remains elusive. Therefore, we examined the expression of Mtp18 upon H2O2 exposure and observed that H2O2 up-regulated Mtp18 expression in a dose- (Figure 2C,E) and time-dependent manner (Figure 2D,F), suggesting that Mtp18 may be involved in the regulation of H2O2-induced mitochondrial fission and apoptosis in HL-1 cells.

H2O2-induced mitochondrial fission is associated with up-regulation in Mtp18 expression

Figure 2
H2O2-induced mitochondrial fission is associated with up-regulation in Mtp18 expression

(A,B) H2O2 exposure induces mitochondrial fission in HL-1 cells. Cells were stimulated with H2O2 at the indicated time points, and mitochondrial morphology was analyzed. (A) Shows mitochondrial morphology. Bar = 5 µm. (B) Shows the percentage of cells undergoing mitochondrial fission. Data were expressed as the mean ± S.E.M. of three independent experiments. (C–F) H2O2 exposure up-regulates the expression of mitochondrial protein 18 (Mtp18) in a dose-and time-dependent manner. Analysis of Mtp18 expression by immunoblot (C,D) and by densitometry (E,F). HL-1 cells were stimulated with the indicated doses of H2O2 and harvested at 24 h (C,E), and cells were stimulated with 200 µmol/l H2O2 and then harvested at the indicated time points (D,F) for immunoblotting. β-actin served as a loading control. The densitometry data were expressed as the mean ± S.E.M. of three independent experiments (E,F). Figures presented are the representative figures of at least three independent experiments. **P<0.01, and ****P<0.0001 vs 0 h.

Figure 2
H2O2-induced mitochondrial fission is associated with up-regulation in Mtp18 expression

(A,B) H2O2 exposure induces mitochondrial fission in HL-1 cells. Cells were stimulated with H2O2 at the indicated time points, and mitochondrial morphology was analyzed. (A) Shows mitochondrial morphology. Bar = 5 µm. (B) Shows the percentage of cells undergoing mitochondrial fission. Data were expressed as the mean ± S.E.M. of three independent experiments. (C–F) H2O2 exposure up-regulates the expression of mitochondrial protein 18 (Mtp18) in a dose-and time-dependent manner. Analysis of Mtp18 expression by immunoblot (C,D) and by densitometry (E,F). HL-1 cells were stimulated with the indicated doses of H2O2 and harvested at 24 h (C,E), and cells were stimulated with 200 µmol/l H2O2 and then harvested at the indicated time points (D,F) for immunoblotting. β-actin served as a loading control. The densitometry data were expressed as the mean ± S.E.M. of three independent experiments (E,F). Figures presented are the representative figures of at least three independent experiments. **P<0.01, and ****P<0.0001 vs 0 h.

Knockdown of Mtp18 reduces hydrogen peroxide-induced mitochondrial fission and apoptosis

To understand the importance of Mtp18 in H2O2-induced cardiotoxicity, we first knocked down the endogenous Mtp18 expression using Mtp18-shRNA. The expression level of Mtp18 was significantly reduced by its shRNA but not by its scrambled control (Figure 3A). We then tested whether Mtp18 is related to the occurrence mitochondrial fission. In the knockdown group, the mitochondria were long thin filamentous, as compared with those showing the punctate structure in the untransfected or scramble-shRNA control groups (Figure 3B). Quantitatively, we found that knockdown of Mtp18 significantly reduced the percentage of cells undergoing mitochondrial fission when compared with its negative or scramble controls at a high concentration of H2O2 exposure (Figure 3C). At the same time, we detected the change in the extent of H2O2-induced apoptosis after knocking down of Mtp18 using TUNEL assays and cell death ELISA. We observed a significant reduction in H2O2-induced TUNEL positive cells and DNA fragmentation in the Mtp18 knockdown group as compared with its scrambled control under high concentration of H2O2 exposure (Figure 3D,E). These data suggest that Mtp18 is essential for mitochondrial fission and initiation of apoptosis in H2O2-induced cardiotoxicity.

Knockdown of Mtp18 reduces H2O2-induced mitochondrial fission and apoptosis

Figure 3
Knockdown of Mtp18 reduces H2O2-induced mitochondrial fission and apoptosis

(A) Analysis of mitochondrial protein 18 (Mtp18) expression. Immunoblot shows Mtp18 knockdown in HL-1 cells (up panel). β-actin served as a loading control. The densitometry data were expressed as the mean ± S.E.M. of three independent experiments (low panel). (B) Shows mitochondrial morphology. (C) Shows the percentage of cells with mitochondrial fission. Cells were exposed to a higher concentration of H2O2, and mitochondria were stained with MitoTraker Red (B,C). (D) Shows the percentage of TUNEL positive cells. (E) Shows apoptosis-related DNA fragmentation. Cells were exposed to H2O2 and apoptosis was analyzed by TUNEL assay (D), and DNA fragments were analyzed using the cell death detection ELISA (E). Data were expressed as the mean ± S.E.M. of three independent experiments. Figures presented are the representative figures of at least three independent experiments. ***P<0.001 and ****P<0.0001.

Figure 3
Knockdown of Mtp18 reduces H2O2-induced mitochondrial fission and apoptosis

(A) Analysis of mitochondrial protein 18 (Mtp18) expression. Immunoblot shows Mtp18 knockdown in HL-1 cells (up panel). β-actin served as a loading control. The densitometry data were expressed as the mean ± S.E.M. of three independent experiments (low panel). (B) Shows mitochondrial morphology. (C) Shows the percentage of cells with mitochondrial fission. Cells were exposed to a higher concentration of H2O2, and mitochondria were stained with MitoTraker Red (B,C). (D) Shows the percentage of TUNEL positive cells. (E) Shows apoptosis-related DNA fragmentation. Cells were exposed to H2O2 and apoptosis was analyzed by TUNEL assay (D), and DNA fragments were analyzed using the cell death detection ELISA (E). Data were expressed as the mean ± S.E.M. of three independent experiments. Figures presented are the representative figures of at least three independent experiments. ***P<0.001 and ****P<0.0001.

Overexpression of Mtp18 sensitizes HL-1 cells to H2O2-induced mitochondrial fission and apoptosis

To test the hypothesis that Mtp18 is pro-mitochondrial fission and pro-apoptosis, we used a sub-cytotoxic dose of H2O2 and asked whether overexpression of Mtp18 is able to increase the sensitivity of cardiac myocytes to H2O2 induced mitochondrial fission and apoptosis. To this end, we infected the cells with the lentiviral construct of Mtp18-cDNA to induce Mtp18 expression (Figure 4A). As observed in Figure 4B, the Mtp18 overexpressed cells show segmented mitochondrial as compared with negative and empty vector control. After 24 h, the cells were treated with a sub-cytotoxic dose of H2O2 (50 µmol/l) and then the percentages of cells with mitochondrial fission and apoptosis were analyzed. We found that a low concentration H2O2 can induce a significantly higher number of cells to undergo mitochondrial fission in the overexpressed group, whereas no statistically significant change in the percentage of mitochondrial fission was noted in the negative and empty vector control groups (Figure 4C). Concomitantly, the suboptimal concentration of H2O2 can induce a significantly higher percentage of apoptosis in the overexpressed group relative to its negative and empty vector controls (Figure 4D,E).

Overexpression of Mtp18 sensitizes HL-1 cells to H2O2-induced mitochondrial fission and apoptosis

Figure 4
Overexpression of Mtp18 sensitizes HL-1 cells to H2O2-induced mitochondrial fission and apoptosis

(A) Analysis of Mtp18 expression. Immunoblot shows Mtp18 overexpression in HL-1 cells (up panel). β-actin served as a loading control. The densitometry data were expressed as the mean ± S.E.M. of three independent experiments (low panel). (B) Shows mitochondrial morphology. Bar = 5 µm. Cells were infected with the lentiviral construct of Mtp18-cDNA to induce Mtp18 expression and empty vector as a control. (C) Enforced expression of Mtp18 sensitizes cells to undergo H2O2-induced mitochondrial fission. HL-1 cells were exposed to a lower concentration (50 µmol/l) of H2O2 for 6 h. (C) Shows the percentage of cells with mitochondrial fission. (D,E) Enforced expression of Mtp18 sensitizes cells to undergo H2O2-induced apoptosis. Cells were exposed to a lower concentration of H2O2, and percentages of apoptosis were analyzed by TUNEL assay (D), and DNA fragments were analyzed using the cell death detection ELISA (E). Data were expressed as the mean ± S.E.M. of three independent experiments. Figures presented are the representative figures of at least three independent experiments. ***P<0.001 and ****P<0.0001.

Figure 4
Overexpression of Mtp18 sensitizes HL-1 cells to H2O2-induced mitochondrial fission and apoptosis

(A) Analysis of Mtp18 expression. Immunoblot shows Mtp18 overexpression in HL-1 cells (up panel). β-actin served as a loading control. The densitometry data were expressed as the mean ± S.E.M. of three independent experiments (low panel). (B) Shows mitochondrial morphology. Bar = 5 µm. Cells were infected with the lentiviral construct of Mtp18-cDNA to induce Mtp18 expression and empty vector as a control. (C) Enforced expression of Mtp18 sensitizes cells to undergo H2O2-induced mitochondrial fission. HL-1 cells were exposed to a lower concentration (50 µmol/l) of H2O2 for 6 h. (C) Shows the percentage of cells with mitochondrial fission. (D,E) Enforced expression of Mtp18 sensitizes cells to undergo H2O2-induced apoptosis. Cells were exposed to a lower concentration of H2O2, and percentages of apoptosis were analyzed by TUNEL assay (D), and DNA fragments were analyzed using the cell death detection ELISA (E). Data were expressed as the mean ± S.E.M. of three independent experiments. Figures presented are the representative figures of at least three independent experiments. ***P<0.001 and ****P<0.0001.

Knockdown of Mtp18 protects the primary cardiomyocytes from undergoing mitochondrial fission and apoptosis upon oxidative stress stimulation

To determine whether the knocking down of Mtp18 protects the cardiomyocytes against oxidative stress injury, we used the primary cardiomyocytes from neonatal rats. Our data showed that H2O2 can induce mitochondrial fission (Figure 5A) and apoptosis observed by TUNEL and DNA fragmentation assay. (Figure 5B). Then, we used lentiviral Mtp18-shRNA to knockdown endogenous Mtp18 expression as described in Figure 5C and the changes in mitochondrial morphology and apoptosis upon hydrogen peroxide exposure were analyzed. As expected, compared with negative control and scrambled groups, the cardiomyocytes infected with Mtp18-shRNA were found to be resistance to mitochondrial fission (Figure 5D,E) and apoptosis under high concentration of H2O2 stimulation (Figure 5F). Therefore, these findings support the protective effect mediated by Mtp18 knockdown during oxidative stress stimulation.

Knockdown of Mtp18 protects the primary cardiomyocytes from undergoing mitochondrial fission and apoptosis upon oxidative stress stimulation

Figure 5
Knockdown of Mtp18 protects the primary cardiomyocytes from undergoing mitochondrial fission and apoptosis upon oxidative stress stimulation

(A) Morphological alteration of neonatal rat cardiomyocytes in response to H2O2 treatment. The cells were treated with 200 µmol/l H2O2 for 6 h. Representative EM images of cardiomyocytes are shown. The arrow indicates fragmented mitochondria. (B) H2O2 induces apoptosis in neonatal rat cardiomyocytes. The cells were treated with 200 µmol/l H2O2 at the indicated time points. Analysis of apoptosis by TUNEL (left panel) and cell death ELISA (Roche, right panel). (C) Mtp18-shRNA significantly decreases Mtp18 expression. Cardiomyocytes were infected with lentiviral Mtp18-shRNA or its scrambled form (Mtp18-sc). Analysis of Mtp18 expression by immunoblot (up panel). The densitometry analysis (low panel). (D) Shows mitochondrial morphology. Bar = 5 µm. (E) Shows the percentage of cells with mitochondrial fission. Cardiomyocytes were infected with lentiviral Mtp18- shRNA as described in (C). After 24 h of infection, cells were treated with H2O2 as in A. The disintegration of mitochondria (mitochondrial fission) in 250–300 cells were counted in 30–40 random fields using confocal microscopy (D and E). (F) Analysis of apoptosis by cell death ELISA (Roche). Cardiomyocytes were infected with lentiviral Mtp18-shRNA as in (C). After 24 h of infection, cells were treated with H2O2. Data are expressed as the mean + S.E.M. of three independent experiments. ***P<0.001 and ****P<0.001. (G) H2O2 induces Drp1 accumulation in mitochondria. H2O2 exposure up-regulates dynamic-related protein 1 (Drp1) expression in whole cell lysate. Cardiomyocytes were treated with H2O2 and then harvested at the indicated time points for immunoblot analysis. Cytochrome c oxidase (Cox4) served as a loading control for HM (heavy membrane pallet, mitochondria) and β-actin served as a loading control for whole cell lysate and cytosolic fraction. Figures presented are the representative figures of at least three independent experiments.

Figure 5
Knockdown of Mtp18 protects the primary cardiomyocytes from undergoing mitochondrial fission and apoptosis upon oxidative stress stimulation

(A) Morphological alteration of neonatal rat cardiomyocytes in response to H2O2 treatment. The cells were treated with 200 µmol/l H2O2 for 6 h. Representative EM images of cardiomyocytes are shown. The arrow indicates fragmented mitochondria. (B) H2O2 induces apoptosis in neonatal rat cardiomyocytes. The cells were treated with 200 µmol/l H2O2 at the indicated time points. Analysis of apoptosis by TUNEL (left panel) and cell death ELISA (Roche, right panel). (C) Mtp18-shRNA significantly decreases Mtp18 expression. Cardiomyocytes were infected with lentiviral Mtp18-shRNA or its scrambled form (Mtp18-sc). Analysis of Mtp18 expression by immunoblot (up panel). The densitometry analysis (low panel). (D) Shows mitochondrial morphology. Bar = 5 µm. (E) Shows the percentage of cells with mitochondrial fission. Cardiomyocytes were infected with lentiviral Mtp18- shRNA as described in (C). After 24 h of infection, cells were treated with H2O2 as in A. The disintegration of mitochondria (mitochondrial fission) in 250–300 cells were counted in 30–40 random fields using confocal microscopy (D and E). (F) Analysis of apoptosis by cell death ELISA (Roche). Cardiomyocytes were infected with lentiviral Mtp18-shRNA as in (C). After 24 h of infection, cells were treated with H2O2. Data are expressed as the mean + S.E.M. of three independent experiments. ***P<0.001 and ****P<0.001. (G) H2O2 induces Drp1 accumulation in mitochondria. H2O2 exposure up-regulates dynamic-related protein 1 (Drp1) expression in whole cell lysate. Cardiomyocytes were treated with H2O2 and then harvested at the indicated time points for immunoblot analysis. Cytochrome c oxidase (Cox4) served as a loading control for HM (heavy membrane pallet, mitochondria) and β-actin served as a loading control for whole cell lysate and cytosolic fraction. Figures presented are the representative figures of at least three independent experiments.

The Mtp18’s target protein network

To understand the molecular mechanism of Mtp18, we performed protein interaction analysis to predict the target protein exhibiting the highest possible functional correlation with Mtp18 [34]. Mtp18’s protein interaction network was analyzed by STRING database. The Mtp18’s functional partners are predicted to be enriched in mitochondrial fission, mitochondrial protein localization, and mitochondrial organization pathways, and are mainly localized in mitochondrial membrane (Table 1). A total of ten potential targets obtained a medium or high confidence score of interaction (≥0.400, Table 2) with H2O2.

Table 1
List of the Gene Ontology biological process and cellular component enriched for proteins present in the STRING protein network
Pathway ID Pathway description Count in gene set False discovery rate 
Biological process (GO) 
GO:0000266 Mitochondrial fission 0.00131 
GO:0070585 Protein localization to mitochondrion 0.00758 
GO:0007005 Mitochondrion organization 0.0262 
GO:0016559 Peroxisome fission 0.0262 
GO:0043653 Mitochondrial fragmentation involved in apoptotic process 0.0262 
Cellular component (GO) 
GO:0005739 Mitochondrion 0.00257 
GO:0005741 Mitochondrial outer membrane 0.016 
GO:0031306 Intrinsic component of mitochondrial outer membrane 0.016 
GO:0031966 Mitochondrial membrane 0.0196 
GO:0005740 Mitochondrial envelope 0.0212 
Pathway ID Pathway description Count in gene set False discovery rate 
Biological process (GO) 
GO:0000266 Mitochondrial fission 0.00131 
GO:0070585 Protein localization to mitochondrion 0.00758 
GO:0007005 Mitochondrion organization 0.0262 
GO:0016559 Peroxisome fission 0.0262 
GO:0043653 Mitochondrial fragmentation involved in apoptotic process 0.0262 
Cellular component (GO) 
GO:0005739 Mitochondrion 0.00257 
GO:0005741 Mitochondrial outer membrane 0.016 
GO:0031306 Intrinsic component of mitochondrial outer membrane 0.016 
GO:0031966 Mitochondrial membrane 0.0196 
GO:0005740 Mitochondrial envelope 0.0212 

The protein interaction network was created using the STRING databases (STRING v10: http://string.embl.de/). The Network includes ten nodes of differentially expressed proteins. Network analysis was set to medium confidence (score > 0.4). The significant was determined using the false discovery rate correction (P<0.05).

Table 2
Predicted functional partners of Mtp18
Protein Name and function Co-expression Text mining Score 
L2hgdh L-2-hydroxyglutarate dehydrogenase √ √ 0.570 
Dnm1l Dynamin 1-like, functions in mitochondrial and peroxisomal division  √ 0.565 
Mfn2 Mitofusin 2 √ √ 0.564 
Dhrs11 Dehydrogenase/reductase (SDR family) member 11  √ 0.558 
Macrod1 MACRO domain containing 1, plays a role in estrogen signaling √ √ 0.547 
Fis1 Fission 1, promotes the fragmentation of the mitochondrial network and its perinuclear clustering  √ 0.524 
Fam195a Family with sequence similarity 195, member A √  0.499 
C78988 RIKEN cDNA 9430016H08 gene  √ 0.495 
Fam185a Family with sequence similarity 185, member A √ √ 0.492 
Apobec2 Apolipoprotein B mRNA editing enzyme, catalytic polypeptide 2  √ 0.479 
Protein Name and function Co-expression Text mining Score 
L2hgdh L-2-hydroxyglutarate dehydrogenase √ √ 0.570 
Dnm1l Dynamin 1-like, functions in mitochondrial and peroxisomal division  √ 0.565 
Mfn2 Mitofusin 2 √ √ 0.564 
Dhrs11 Dehydrogenase/reductase (SDR family) member 11  √ 0.558 
Macrod1 MACRO domain containing 1, plays a role in estrogen signaling √ √ 0.547 
Fis1 Fission 1, promotes the fragmentation of the mitochondrial network and its perinuclear clustering  √ 0.524 
Fam195a Family with sequence similarity 195, member A √  0.499 
C78988 RIKEN cDNA 9430016H08 gene  √ 0.495 
Fam185a Family with sequence similarity 185, member A √ √ 0.492 
Apobec2 Apolipoprotein B mRNA editing enzyme, catalytic polypeptide 2  √ 0.479 

The Mtp8’s functional partners were predicted using string v10 (http://string-db.org/cgi/input.pl). The confidence interaction scores range from 0.4–0.6. The evidence for the calculation of interaction scores was obtained from co-expression and/or text mining result.

Of which, L-2-hydroxydehydroxy-glutarate dehydrogenase (L2hgdh) scores the highest followed by Drp1 and Mitofusin2 (Mfn2). Since L2hgdh has not yet been reported to play a role in the regulation of mitochondrial dynamics, we decided to study the functional correlation with the second highest predicted protein, Drp1. Drp1 has been reported to be essential for the initiation of mitochondrial fission, and the functional correlation between Mtp18 and Drp1 was previously reported in cancer cell lines [16,22,35]. Therefore, we decided to study the mechanism of interaction between Mtp18 and Drp1.

Mtp18 enhances hydrogen peroxide-induced Drp1 accumulation in mitochondria

To understand the role of Drp1 in the regulation of H2O2-induced apoptosis, we performed subcellular fraction and compared the expression levels of Drp1 between cytosolic and mitochondrial compartment before and after H2O2 exposure. Drp1 was up-regulated upon H2O2 exposure in whole cell lysate (Figure 6A–D, upper panels). On subcellular fraction analysis, we observed that Drp1 was translocated from cytosol to mitochondria upon H2O2 treatment (Figure 6A–D, middle and lower panels). In primary cardiomyocytes, we also confirmed the translocation of cytosolic Drp1 to mitochondria (Figure 5G) in response to H2O2 treatment.

Hydrogen peroxide exposure in HL-1 cells induces recruitment of Drp1 in mitochondria

Figure 6
Hydrogen peroxide exposure in HL-1 cells induces recruitment of Drp1 in mitochondria

(A,C) Show Drp1 expression by immunoblot, and (B,D) show the densitometry. HL-1 cells were stimulated with the indicated doses of H2O2, harvested at 24 h (A and B), and treated with H2O2 and then harvested at the indicated time points (C and D) for immunoblotting. β-actin served as a loading control for whole cell lysate and cytosolic components, and cytochrome c oxidase (Cox4) for mitochondrial fraction. The displayed images were cropped from the original blot. The densitometry data were expressed as the mean ± S.E.M. of three independent experiments (B and D). Figures presented are the representative figures of at least three independent experiments. **P<0.01, ***P<0.001, and ****P<0.0001 vs 0 h.

HM: mitochondrial enriched heavy membrane fraction.

Figure 6
Hydrogen peroxide exposure in HL-1 cells induces recruitment of Drp1 in mitochondria

(A,C) Show Drp1 expression by immunoblot, and (B,D) show the densitometry. HL-1 cells were stimulated with the indicated doses of H2O2, harvested at 24 h (A and B), and treated with H2O2 and then harvested at the indicated time points (C and D) for immunoblotting. β-actin served as a loading control for whole cell lysate and cytosolic components, and cytochrome c oxidase (Cox4) for mitochondrial fraction. The displayed images were cropped from the original blot. The densitometry data were expressed as the mean ± S.E.M. of three independent experiments (B and D). Figures presented are the representative figures of at least three independent experiments. **P<0.01, ***P<0.001, and ****P<0.0001 vs 0 h.

HM: mitochondrial enriched heavy membrane fraction.

To further understand whether Mtp18 can influence Drp1 translocation upon H2O2 exposure, we tested whether Mtp18 knockdown can affect the Drp1 recruitment in mitochondria. Our data showed that knockdown of Mtp18 prevents the Drp1 translocation into mitochondria even under high dose H2O2 exposure (Figure 7A). These data indicated that Mtp18 is required for Drp1 accumulation in mitochondria.

Mtp18 enhances H2O2-induced Drp1 accumulation in mitochondria

Figure 7
Mtp18 enhances H2O2-induced Drp1 accumulation in mitochondria

(A) Knockdown of Mtp18 inhibits H2O2-induced Drp1 accumulation in mitochondria. (A) Shows Drp1 expression in subcellular fraction. β-actin served as a loading control for the cytosolic component. Cox4 served as a loading control for the mitochondrial component. HM = mitochondria-enriched HMs. Figures presented are the representative figures of at least three independent experiments. (B) Knockdown of Drp1 decreases H2O2-induced apoptosis. Cells were transfected with scrambled siRNA or Drp1 siRNA (r) respectively for 24 h. Then, they were treated with a higher concentration of H2O2 for 6 h, and DNA fragmentation was analyzed using the cell death detection ELISA. (C) Mtp18 and Drp1 are interdependent in mediating apoptosis. Overexpression of Mtp18 or Drp1 was performed by infecting with lentivirus overexpressing Mtp18 or Drp1. Knockdown of Mtp18 and Drp1 were performed using Mtp18-shRNA or Drp1-siRNA, respectively. After infecting the cells with corresponding overexpression or knockdown plasmids, cells were exposed to H2O2, and DNA fragmentation was analyzed using the cell death detection ELISA. (B,C) Show DNA fragmentation. Data were expressed as the mean ± S.E.M. of three independent experiments. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

Figure 7
Mtp18 enhances H2O2-induced Drp1 accumulation in mitochondria

(A) Knockdown of Mtp18 inhibits H2O2-induced Drp1 accumulation in mitochondria. (A) Shows Drp1 expression in subcellular fraction. β-actin served as a loading control for the cytosolic component. Cox4 served as a loading control for the mitochondrial component. HM = mitochondria-enriched HMs. Figures presented are the representative figures of at least three independent experiments. (B) Knockdown of Drp1 decreases H2O2-induced apoptosis. Cells were transfected with scrambled siRNA or Drp1 siRNA (r) respectively for 24 h. Then, they were treated with a higher concentration of H2O2 for 6 h, and DNA fragmentation was analyzed using the cell death detection ELISA. (C) Mtp18 and Drp1 are interdependent in mediating apoptosis. Overexpression of Mtp18 or Drp1 was performed by infecting with lentivirus overexpressing Mtp18 or Drp1. Knockdown of Mtp18 and Drp1 were performed using Mtp18-shRNA or Drp1-siRNA, respectively. After infecting the cells with corresponding overexpression or knockdown plasmids, cells were exposed to H2O2, and DNA fragmentation was analyzed using the cell death detection ELISA. (B,C) Show DNA fragmentation. Data were expressed as the mean ± S.E.M. of three independent experiments. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

Mtp18 and Drp1 are interdependent in mediating apoptosis

We then tested whether Drp1 is required for H2O2-induced apoptosis by knocking down the endogenous Drp1 expression using Drp1-siRNA and exposed the cells to high concentration of H2O2. We observed that knockdown of Drp1 can prevent H2O2-induced mitochondrial fission and apoptosis compared with its scrambled and negative control (Supplementary Figure S2 and Figure 7B). Then we wanted to understand whether Mtp18 is dependent on Drp1 to exert its pro-apoptotic effect. To this end, we overexpressed Mtp18 or Drp1 using the Lenti-ORF clone of Mtp18 cDNA or Drp1 cDNA, respectively. We then knockdown Drp1 in the presence of Mtp18 overexpression and exposed the cells to H2O2. Alternatively, we knocked down Mtp18 in the presence of Drp1 overexpression and exposed the cells to H2O2. As shown in Figure 7C, we found that the functions of these two proteins are interdependent. Overexpression of Mtp18 could not induce significant apoptosis if Drp1 is minimally expressed and vice versa. Further, we found that overexpression of Mtp18 increases Drp1 phosphorylation upon H2O2 treatment (Supplementary Figure S3A–D). Collectively, these findings indicate the importance of Mtp18 and Drp1 in H2O2-induced apoptosis.

Discussion

Growing bodies of evidence have indicated that apoptosis plays a pivotal role in the progression of a variety cardiovascular diseases. Although apoptosis is not the only mode of cell death in the cardiovascular system, both in vivo and in vitro studies have evidenced that inhibition of apoptosis can prevent the heart from failing and maintain its function to a considerable extent [36,37]. Therefore, a thorough understanding of the molecular mechanism of apoptosis can provide novel therapeutic insights into protection of the dying heart. Here, we observed that Mtp18 could regulate mitochondrial fission and apoptosis in both HL-1 cardiac myocytes and primary cardiomyocytes during oxidative stress stimulation by targetting Drp1-mediated fission.

Mitochondrial morphology is dynamic and interchangeable between fusion (elongated interconnected mitochondrial networks) and fission (fragmented, disconnected arrangement) depending on various physiological and pathological stimuli. Under normal physiology, mitochondria tend to fuse to distribute ATP throughout the cells, whereas during cell division, mitochondria favor division to allow correct distribution of mitochondrial DNA. Alteration in mitochondrial morphology is associated with various cardiovascular abnormalities [26]. Mitochondrial dynamics is controlled by the complex interactions among fusion and fission proteins [26,38].

Drp1 is mainly cytosolic and can be recruited to scission sites on the outer mitochondrial membrane (OMM) to induce fission. The Drp1 translocation to OMM is a pre-requisite for mitochondrial fission and initiation of apoptosis [11]. Structurally, Drp1 composes of three domains including GTPase, the central domain, and the GTPase effector domain (GED). Drp1 recruitment or asS.E.M.bly is a post-translational mechanism and is being regulated by sumoylation, ubiquitination [39], S- nitrosylation [40], or phosphorylation of serine residues [41,42] within the GED. Several proteins, such as Fis1, Mid49, Mid51, and MFF, are involved in Drp1 recruitment in OMM [43,44]. Fis1 can function as a receptor protein for Drp1 in mitochondria [45]. However, a recent study conducted in cancer cell lines have shown that either hFis1 or Drp1 overexpression by themselves are not sufficient to induce mitochondrial fission if Mtp18 is lacking [18,46,47]. Similarly, our findings in HL-1 cells and primary cardiomyocytes showed that Mtp18 targetted Drp1 in cardiomyocyte mitochondrial fission event. We found that H2O2-induced Drp1 recruitment in OMM was impaired when Mtp18 was minimally expressed.

Mtp18 is a small protein of 18 kDa and composed of 166 amino acids. It is thought to be a transmitochondrial inner membrane protein, exposing mainly to mitochondrial intermembrane space. It was first identified in 2004 as a novel downstream effector of PI 3-kinase signaling [19]. The C-terminus of Mtp18 is responsible for mitochondrial fragmentation. Mtp18 is highly expressed in muscle and heart [18,19]. However, the role of Mtp18 in cardiomyocytes remains elusive. Previous studies in different cancer cell lines indicate that the effect of Mtp18 in mitochondrial fission might be cell type specific. In human keratinocyte (HeCaT) and human prostate cancer (PC-3) cells, reduction of Mtp18 expression results in mitochondrial fission, and a dramatic decrease in cell proliferation as well as a significant increase in DNA fragmentation, indicating that Mtp18 is essential for mitochondrial fusion and normal cell growth [19]. By contrast, in human cervical epithelial cancer (HeLa) cells, transient knocked down of Mtp18 expression with shRNA give rise to long thin filamentous mitochondria indicating that Mtp18 is required for mitochondrial fission. Having the controversy of Mtp18’s role in mitochondrial fission machinery, there was loosen of track on Mtp18’s publication since 2005. Consistent with the findings in HeLa cell, our recent report in gastric cancer cells and HL-1 cardiac myocytes revealed that Mtp18 could promote chemotherapy-induced apoptosis.

In the current study, we found that Mtp18 expression is up-regulated during H2O2 exposure and Mtp18 promotes mitochondrial fission and apoptosis in HL-1 cells and neonatal rat cardiomyocytes. Accordingly, reduction of Mtp18 expression inhibits H2O2-induced mitochondrial fission and significantly reduces apoptosis. Mtp18 participates in H2O2-induced Drp1 translocation. Mechanistically, we found that knockdown of Mtp18 interferes Drp1 accumulation in OMM and prevents the fission of mitochondrial membrane. These findings suggest that Mtp18-induced mitochondrial fission is mediated through Drp1. However, we fail to indicate whether Mtp18-Drp1 interaction is either directly or bridged by other fission proteins. Questions as to whether Mtp18 can influence Drp1 activity in terms of sumoylation, ubiquitination, or phosphorylation remain to be elucidated. Another interesting question is why the trends of Mtp18 expression in response to oxidative stress show an opposite direction in cancer and cardiomyocytes. This highlights that the regulatory mechanism of Mtp18 expression in different cell lines could be varied. And thus, it is imperative to understand the molecular mechanism by which Mtp18 expression is regulated.

Conclusion

In summary, our current work reveals that Mtp18 plays a crucial role in regulating cardiomyocyte apoptosis. Mtp18 promotes H2O2-induced apoptosis by enhancing Drp1-mediated mitochondrial fission. The signaling pathway proposed in the present study is summarized in Figure 8. Our data convince that knockdown of Mtp18 can minimize H2O2-induced cardiomyocyte loss by interfering Drp1 recruitment in OMM. Henceforward, the present study provides a novel therapeutic insight to minimize cardiomyocyte loss during oxidative-stress heart injury.

A schematic description of the relationship between Mtp18 and Drp1 in cardiomyocytes

Figure 8
A schematic description of the relationship between Mtp18 and Drp1 in cardiomyocytes

H2O2 stimulation up-regulates Mtp18 and Drp1 expression. Mtp18 promotes Drp1 phosphorylation and thereby enhancing Drp1 accumulation in mitochondria. Mtp18 could mediate mitochondrial fission by serving as an inner mitochondrial membrane component to harness Drp1 accumulation and thus further activates apoptosis. Under normal physiology, the cells are protected against apoptosis by expressing less Mtp18 and Drp1.

Figure 8
A schematic description of the relationship between Mtp18 and Drp1 in cardiomyocytes

H2O2 stimulation up-regulates Mtp18 and Drp1 expression. Mtp18 promotes Drp1 phosphorylation and thereby enhancing Drp1 accumulation in mitochondria. Mtp18 could mediate mitochondrial fission by serving as an inner mitochondrial membrane component to harness Drp1 accumulation and thus further activates apoptosis. Under normal physiology, the cells are protected against apoptosis by expressing less Mtp18 and Drp1.

Clinical perspectives

  • Long-term ROS accumulation in the cardiovascular system is seen in cardiac stress conditions including coronary artery disease, aging-related cardiovascular abnormalities, and exposure to cardiac stressors such as hydrogen peroxide, and doxorubicin.

  • Mtp18, a cardiac abundant mitochondrial inner membrane protein, could regulate oxidative stress-mediated mitochondrial fission and apoptosis in cardiomyocytes.

  • We found that knockdown of Mtp18 interfered with Drp1-associated mitochondrial fission and subsequent activation of apoptosis in both HL-1 cells and primary cardiomyocytes. The present study highlights the role of Mtp18 in cardiac apoptosis and provide a novel therapeutic insight to minimize cardiomyocyte loss via targetting mitochondrial dynamics.

Acknowledgements

We thank Dr. Xing Rong from Children’s Heart Center, the Second Affiliated Hospital, and Yuying Children’s Hospital, Institute of Cardiovascular Development and Translational Medicine, Wenzhou Medical University, Zhejiang, China for providing us HL-1 cell line.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by National Natural Science Foundation of China [grant numbers 81741173, 31430041, and 81470437] and National Natural Science Foundation of China Research Fund for International Young Scientists [grant number 81850410551].

Author Contribution

P.L. and L.H.H.A. conceived and designed the study. L.H.H.A., Y.Z.L., H.Y., X.C., Z.Y., and J.G. performed the experiments. L.H.H.A. and P.L. analyzed the data, wrote, edited, and submitted the manuscript. All authors discussed and finalized the manuscript for submission.

Abbreviations

     
  • Cyt-c

    cytochrome-c

  •  
  • Drp1

    dynamin-related protein 1

  •  
  • EM

    electron microscopy

  •  
  • FBS

    fetal bovine serum

  •  
  • GED

    GTPase effector domain

  •  
  • GO

    Gene Ontology

  •  
  • H2O2

    hydrogen peroxide

  •  
  • HeLa

    human cervical epithelial cancer

  •  
  • OMM

    outer mitochondrial membrane

  •  
  • PARP

    poly ADP ribose polymerase

  •  
  • ROS

    reactive oxygen species

  •  
  • RT

    room temperature

  •  
  • TUNEL

    terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling

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Author notes

*

These authors equally contributed to this work

Supplementary data