Altered cardiac mitochondrial dynamics with excessive fission is a predominant cause of cardiac dysfunction during ischemia/reperfusion (I/R) injury. Although pre-ischemic inhibition of mitochondrial fission has been shown to improve cardiac function in I/R injury, the effects of this inhibitor given at different time-points during cardiac I/R injury are unknown. Fifty male Wistar rats were subjected to sham and cardiac I/R injury. For cardiac I/R injury, rats were randomly divided into pre-ischemia, during-ischemia, and upon onset of reperfusion group. A mitochondrial fission inhibitor, Mdivi-1 (mitochondrial division inhibitor 1) (1.2 mg/kg) was used. During I/R protocols, the left ventricular (LV) function, arrhythmia score, and mortality rate were determined. Then, the heart was removed to determine infarct size, mitochondrial function, mitochondrial dynamics, and apoptosis. Our results showed that Mdivi-1 given prior to ischemia, exerted the highest level of cardioprotection quantitated through the attenuated incidence of arrhythmia, reduced infarct size, improved cardiac mitochondrial function and fragmentation, and decreased cardiac apoptosis, leading to preserved LV function during I/R injury. Mdivi-1 administered during ischemia and upon the onset of reperfusion also improved cardiac mitochondrial function and LV function, but at a lower efficacy than when it was given prior to ischemia. Taken together, mitochondrial fission inhibition after myocardial ischemic insults still exerts cardioprotection by attenuating mitochondrial dysfunction and dynamic imbalance, leading to decreased infarct size and ultimately improved LV function after acute cardiac I/R injury in rats. These findings indicate its potential clinical usefulness.

Introduction

Cardiac ischemia/reperfusion (I/R) injury has been extensively studied in past decades [1–3], and several novel pharmacological and device interventions have shown promising results regarding their efficacy in cardioprotection [4–7]. Recently, growing evidence indicates the important relationship between the roles of mitochondrial dynamics and cardiac I/R injury [8–11]. During mitochondrial fission, activated cytosolic Dynamin-related protein 1 (Drp1) binds with its adapter proteins located on the mitochondrial outer membrane, allowing the incision of the mitochondrial membrane and thus initiating the process of mitophagy to remove damaged mitochondria [12]. However, during cardiac I/R injury, the process of mitophagy did not effectively eliminate these excessively damaged mitochondria, leading to the accumulation of excessive mitochondrial fragmentation [13] and also activating apoptotic pathways though the release of cytochrome c (Cyt c) and the activation of caspases [14]. All of these events cause further cellular damage during cardiac I/R.

Since altered mitochondrial dynamics play an important role in cardiac I/R injury, pharmacological strategies to modulate these abnormal processes in mitochondria have been investigated [9,10,15]. By using the inhibitor of mitochondrial fission Mdivi-1 (mitochondrial division inhibitor 1), it has been shown to reduce excessive mitochondrial fission, and prevent cardiomyocyte apoptosis in HL-1 cells under hypoxia-reoxygenation [15–17]. Moreover, Mdivi-1 has been shown to improve cardiac function in isolated rat hearts with global I/R injury [8] as well as in mice [15,18,19]. Furthermore, given Mdivi-1 for five consecutive days (2 days prior to I/R injury, on the day of I/R injury, and 2 days after I/R injury) also improved cardiac function in I/R injury rats [20]. Despite these promising results, these findings are not clinically applicable since in all of these studies, Mdivi-1 was given prior to ischemia, a factor which is irrelevant to real clinical application due to the fact that patients with acute myocardial infarction (AMI) generally seek medical attention after cardiac ischemia has already occurred [11,15]. Ding et al. [21] reported the cardioprotective effects of Mdivi-1 given before reperfusion in cardiac I/R injury of diabetic rats. However, the severity of their model was different from our model since those diabetic rats had an excessive mitochondrial fission. Therefore, further research is essential to investigate the potential therapeutic efficacy of the timing of the administration of this drug in cases of I/R injury.

In this in vivo study, we aimed to investigate the temporal effects of the mitochondrial fission inhibitor, Mdivi-1, given at different time-points including pre-ischemia, during-ischemia, and upon the onset of reperfusion in rats with cardiac I/R injury. Our results demonstrated that inhibition of mitochondrial fission even after myocardial ischemic onset still provided cardioprotective benefits. These promising benefits of mitochondrial fission inhibitor given after the ischemic onset or reperfusion would pave the way for potential therapeutic strategies for AMI patients in the near future.

Experimental

Animal model preparation

All rat experiments conformed to the NIH guidelines (Guide for the Care and Use of Laboratory Animals) and were carried out according to the protocol approved by the Institutional Animal Care and Use Committees of the Faculty of Medicine, Chiang Mai University, Thailand. Male Wistar rats (n=50, ~350 g, 8-week-old) were obtained from the National Laboratory Animal Center, Mahidol Universiry, Bangkok, Thailand. This model of male Wistar rat has been in use from our previous reports [4,7,22]. All animals were maintained in environmentally controlled conditions (25 ± 0.5°C, 12-h light/12-h dark cycle) and fed normal rat chow and water ad libitum for 1 week to allow acclimatization before study.

Experimental design

Rats were randomly divided into sham and cardiac I/R groups. In cardiac I/R, rats were divided into four subgroups to receive different treatments (Figure 1); (i) Pretreated group: received mitochondrial fission inhibitor Mdivi-1 at 15 min before ischemia; (ii) Ischemia group: received Mdivi-1 15 min after the onset of ischemia; (iii) Reperfusion group: received Mdivi-1 at the onset of reperfusion; and (iv) Placebo group: received normal saline as a vehicle. Mdivi-1 (Sigma–Aldrich Co.) 1.2 mg/kg dissolved in 1 ml of 0.9% normal saline solution was administered via intravenous injection through the femoral vein. This dose was chosen since it has been shown to exert cardioprotection in mice [15]. During the I/R protocol, the left ventricular (LV) function was recorded using a pressure-volume (P-V) loop recording device (Transonic Scisense Inc., London, ON, Canada) [7]. A surface electrocardiogram (ECG) was used to record and determine arrhythmia score and the mortality rate. At the end of the I/R protocol, the heart was removed rapidly, following decapitation under deep anesthesia, for infarct size measurement and cardiac tissue studies.

The study protocol

Figure 1
The study protocol

Study protocol of the effects of mitochondrial fission inhibitor on cardiac and mitochondrial function given at pre-ischemia, during ischemia, and onset of reperfusion during acute I/R injury in lean rats; n=10 per group.

Figure 1
The study protocol

Study protocol of the effects of mitochondrial fission inhibitor on cardiac and mitochondrial function given at pre-ischemia, during ischemia, and onset of reperfusion during acute I/R injury in lean rats; n=10 per group.

Surgical preparation of the cardiac I/R injury

Rats were anesthetized using Zoletil (50 mg/kg, Virbac Laboratories, Carros, France) and Xylazine (0.15 mg/kg, Laboratorios Calier S.A., Barcelona, Spain) intramuscular injections. After the rats were anesthetized, the level of anesthesia was determined by monitoring the respiratory rate and effort, color of the mucous membranes, and reflected eye color, pedal reflex (firm toe pinch) and we adjusted the anesthetic delivery appropriately to maintain the surgical plane. After the tracheostomy was done, the rats were ventilated with room air from a positive pressure ventilator (CWE Inc, Ardmore, PA, U.S.A.). Lead II ECG (PowerLab 4/25 T, AD Instruments) was recorded throughout the study. A left-side thoracotomy was performed at the fourth intercostal space, the pericardium was incised and the heart exposed. The left anterior descending (LAD) coronary artery was identified and ligated at ~2 mm distal to its origin with a 5-0 silk suture using a traumatic needle. The end of this ligature was passed through a small vinyl tube, which was used to occlude the coronary artery by pulling the thread. Ischemia was confirmed by an ST segment elevation on the ECG and a change in color of the myocardial tissue around the ischemic area. After 30 min of ischemia, the ligature was loosened, and the ischemic myocardium was continuously reperfused for 2 h [5,23].

Arrhythmia determination

Lead II ECG was used to determine the arrhythmia score. The occurrence of arrhythmia was characterized using the Lambeth Conventions, and arrhythmia score was determined based on the criteria of Curtis et al. [24].

Determination of LV function

The right carotid artery was identified and a P-V catheter (Scisense, Ontario, Canada) was inserted and advanced into the left ventricle to determine LV function during the I/R injury. Heart rate (HR), LV end-systolic pressure (LVESP), LV end-diastolic pressure (LVEDP), ventricular contractility assessment (dP/dt) max, dP/dt min, stroke volume (SV), and LV ejection fraction (LVEF) were determined using an analytical software program (Labscribe, Dover, New Hampshire, U.S.A.) [5,7]. For pressure-volume loop (P-V loop) data analysis, (i) 50 loops before ischemia were selected to represent the baseline, (ii) 50 loops at the end of coronary occlusion were selected to represent the ischemic period, and (iii) 50 loops at the end of reperfusion were selected to represent the reperfusion period.

Myocardial infarct size determination

At the end of the study protocol, the rat was killed by decapitation and the heart was rapidly removed. Then, the LAD was occluded again at the same occlusion site as had been done during I/R. The heart was perfused with 1 ml Evan’s blue dye via the aorta to determine the LV area at risk (AAR). The heart was frozen at −20°C overnight and then sectioned horizontally from the apex to the occlusion site into seven to nine slices. Then, 2,3,5-triphenyltetrazolium chloride (TTC) was added to each slice for 15 min, and the area with viable tissues was seen in red. The infarct size was determined from the area that was not stained with Evan’s blue and TTC. The infarct size and the AAR were determined using the ImageTool software version 3.0 and were calculated according to Reiss et al.’s formula [5,23].

Isolated cardiac mitochondrial studies

At the end of the I/R protocol, the heart was removed, and cardiac mitochondria were isolated from the ischemic (I) and non-ischemic or remote (R) areas of the ventricles as described previously [25]. The protein concentration was determined according to BCA assay. The isolated cardiac mitochondria were used to determine mitochondrial function including mitochondrial reactive oxygen species (ROS) levels, mitochondrial membrane potential change and mitochondrial swelling. Cardiac mitochondrial function was determined as described previously [5]. In brief, the mitochondrial ROS level was measured using a dichlorohydro-fluorescein diacetate dye (DCFDA). DCFDA is oxidized in the presence of H2O2 to a dichlorofluorescein (DCF). The fluorescence intensity of the DCF was measured using a fluorescent microplate reader (excitation at 485 nm and emission at 530 nm). The ROS levels were expressed as arbitrary units of fluorescence intensity of DCF [5].

The change in mitochondrial membrane potential was measured using 5,5′,6,6′-tetrachloro-1,1′,3,3′ -tetraethylbenzimidazolcarbocyanine iodide dye (JC-1). JC-1 is a ratiometric dye which is internalized as a monomer dye (green fluorescence, excitation at 485 nm and emission at 530 nm) and is concentrated by respiring mitochondria with a negative inner membrane potential into a J-aggregate dye (red fluorescence, excitation at 485 nm and emission at 590 nm). The intensity of fluorescence was determined using a fluorescent microplate reader. The change in mitochondrial membrane potential was calculated from the ratio of red to green fluorescence. In the present study, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio [5].

The isolated mitochondrial suspension was used to determine mitochondrial swelling by measuring the change in the absorbance of the mitochondrial suspension detected at 540 nm by using a microplate reader. Mitochondrial swelling was indicated by a decrease in the absorbance of the mitochondrial suspension. In addition, TEM was used to determine the morphology of isolated cardiac mitochondria [5].

Western blot analysis for analysis of mitochondrial dynamics, apoptotic pathway, and Connexin 43

Myocardial protein extracts were divided into ischemic (I) and non-ischemic or remote (R) areas. Protein concentration from the heart tissues was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, U.S.A.). Protein (50–80 μg) was added to the loading buffer. After that, proteins were transferred to nitrocellulose membranes in the presence of a transfer buffer. Membranes were blocked with 5% skimmed milk for 1 h at room temperature. Next, the membranes were exposed overnight to anti-Drp1, anti-Drp1 phosphorylated at Ser616, anti-mitofusin 2 (Mfn2), and anti-optic atrophy 1 (OPA1) to determine cardiac mitochondrial dynamics. In addition, anti-Bax, anti-Bcl2, anti-caspase-3, anti-cleaved caspase-3, anti-Cyt c (Cell Signaling Technology, Danvers, MA, U.S.A.) were used to determine cardiac apoptotic signaling. For cardiac gap junction protein assessment, anti-connexin-43 (Cx43), and anti-Cx43 phosphorylated at Ser368 were used. Subsequently, the membranes were washed and incubated with horseradish peroxidase conjugated with anti-rabbit IgG (Cell Signaling Technology). Finally, bands were detected and used for the analysis of protein expression [5].

Terminal deoxynucleotidyl transferase nick-end labeling positive cells for quantitation of cardiomyocyte apoptosis

Terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay was used to detect cardiomyocyte apoptosis by using an in situ cell death detection kit (Roche, Basel, Switzerland). For in situ labeling, the cardiac tissue slices from ischemic area were placed in 1× PBS for 10 min after dehydration. The samples were covered with 50 µl of Proteinase k solution (1:50) for 30 min followed by 50 µl of Cytonin™ for 120 min. For positive control, the samples were covered with TACS nuclease 1:50 in TACS nuclease buffer. TUNEL-positive cells were detected with fluorescence microscope (Nikon, Tokyo, Japan) at λex 494 nm and λem 512 nm. DAPI was detected at λex 358 nm and λem 461 nm. The apoptosis index was calculated as a percentage of the number of TUNEL-positive apoptotic cells over the total number of nucleated cells (DAPI staining) [26].

Data and statistical analysis, and statistical analysis

The experimental procedures or treatment and data analyses were carried out with randomization and blinding. Data were expressed as mean ± S.E.M. Data were analyzed with GraphPad Prism 7.0 software. A one-way ANOVA followed by an LSD post hoc test were used to test the difference between groups. Mortality rate was analyzed using a Chi-square test. P<0.05 was considered statistically significant.

Results

Mdivi-1 given after myocardial ischemia effectively reduced the infarct size and mortality rate

In the present study, the AAR in the control group (43 ± 1%) was not different from that in any groups treated with mitochondrial fission inhibitor Mdivi-1 (pretreated group (44 ± 0.8%), during ischemia (43 ± 1%), and onset of reperfusion (43 ± 0.6%)). These data indicated that the AAR was not different amongst groups in the present study. However, Mdivi-1 given at different time-points (pretreatment, during-ischemia, and at onset of reperfusion) significantly reduced the myocardial infarction size, compared with the control group (Figure 2A). Differential temporal effects of the drug administration on the infarct size were clearly observed, the infarct size in the pretreated group (~83% reduction compared with the control) being significantly smaller than those in the group being treated during ischemia (~56% reduction) and at the onset of reperfusion (~53% reduction) (Figure 2A). The mortality rate was also significantly decreased in all Mdivi-1 treated groups, compared with the control group (Figure 2B). In-line with the infarct size reduction, rats in the Mdivi-1 pretreated group had a lower mortality rate, compared with the Mdivi-1 treated during ischemia and at onset of reperfusion groups (Figure 2B).

Myocardial infarction size and mortality rate

Figure 2
Myocardial infarction size and mortality rate

The effect of Mdivi-1 given at different time-points (pretreatment, treated during ischemia, and onset of reperfusion) on (A) myocardial infarction size; the blue area = perfused and alive, the red and white areas = AAR; the white area = infarction; scale bar: 5 mm; n=4–5 per group. (B) Mortality rate; n=10 per group. P<0.05 compared with control; P<0.05 compared with pretreated.

Figure 2
Myocardial infarction size and mortality rate

The effect of Mdivi-1 given at different time-points (pretreatment, treated during ischemia, and onset of reperfusion) on (A) myocardial infarction size; the blue area = perfused and alive, the red and white areas = AAR; the white area = infarction; scale bar: 5 mm; n=4–5 per group. (B) Mortality rate; n=10 per group. P<0.05 compared with control; P<0.05 compared with pretreated.

Mdivi-1 reduced cardiac arrhythmia during I/R

The arrhythmia score in the Mdivi-1 pretreated group was significantly decreased, when compared with the control group (Figure 3A). However, Mdivi-1 treatment during ischemia and at the onset of reperfusion did not reduce the arrhythmia score (Figure 3A). In addition to the arrhythmia score, the level of Cx43 phosphorylation (pCx43) was determined. In the control group, the ratio of pCx43 and total Cx43 was significantly decreased, compared with sham group. Interestingly, the level of pCx43 at Ser368 in cardiac tissue was higher in the Mdivi-1 pretreated group, compared with the control group (Figure 3B). However, Mdivi-1 treatment during ischemia and at the onset of reperfusion did not change this level, when compared with the control group (Figure 3B).

Arrhythmia score and the expression of Cx43

Figure 3
Arrhythmia score and the expression of Cx43

The effect of Mdivi-1 given at different time-points (pretreatment, treated during ischemia, and onset of reperfusion) on arrhythmias. (A) Arrhythmias score; n=4–5 per group. (B) pCx43 (Ser368)/total Cx43 from ischemic area; n=4 per group. pCx43 (Ser368) = phosphorylation Cx43 at Ser368. *P<0.05 compared with sham; P<0.05 compared with control; P<0.05 compared with pre-treated.

Figure 3
Arrhythmia score and the expression of Cx43

The effect of Mdivi-1 given at different time-points (pretreatment, treated during ischemia, and onset of reperfusion) on arrhythmias. (A) Arrhythmias score; n=4–5 per group. (B) pCx43 (Ser368)/total Cx43 from ischemic area; n=4 per group. pCx43 (Ser368) = phosphorylation Cx43 at Ser368. *P<0.05 compared with sham; P<0.05 compared with control; P<0.05 compared with pre-treated.

Mdivi-1 improved LV dysfunction during I/R injury

Changes in LV function was shown at different time-points are summarized in Figure 4A–G. At the baseline, there was no significant difference in LV parameters amongst groups. After LAD occlusion, the results showed that Mdivi-1 pretreatment and treatment during ischemia improved SV, LVESP, LVEDP, +dP/dt, and %LVEF (Figure 4B–E,G, respectively). However, during the reperfusion period, all showed an improvement against I/R injury in all Mdivi-1 treated groups. Although our data showed that the cardiac function was improved in all Mdivi-1 treated groups, the LVEF was greatly improved in the pretreatment at reperfusion, compared with the other two treatment groups (Figure 4G).

Cardiac function parameters

Figure 4
Cardiac function parameters

The effect of Mdivi-1 given at different time-points (pretreatment, treated during ischemia, and onset of reperfusion) on LV function. (A) HR. (B) SV. (C) LVESP. (D) LVEDP. (E) +dP/dt max. (F) −dP/dt min. (G) LVEF. n=4–5 per group. *P<0.05 compared with baseline of its group; P<0.05 compared with ischemia of its group; P<0.05 compared with control group at that period; #P<0.05 compared with pretreated group at that period.

Figure 4
Cardiac function parameters

The effect of Mdivi-1 given at different time-points (pretreatment, treated during ischemia, and onset of reperfusion) on LV function. (A) HR. (B) SV. (C) LVESP. (D) LVEDP. (E) +dP/dt max. (F) −dP/dt min. (G) LVEF. n=4–5 per group. *P<0.05 compared with baseline of its group; P<0.05 compared with ischemia of its group; P<0.05 compared with control group at that period; #P<0.05 compared with pretreated group at that period.

Mdivi-1 attenuated cardiac cell apoptosis

Pro-apoptotic protein Bax, anti-apoptotic protein Bcl-2, Cyt c, and cleaved-caspase 3 are shown in Figure 5A–D. The control group with I/R alone was significantly increased Bax, Cyt c, and cleaved-caspase 3 levels, when compared with sham group. According to Mdivi-1 treated groups, the level of Bax expression, only rats in the Mdivi-1 pretreated group had significantly reduced Bax levels, when compared with the control group (Figure 5A). For anti-apoptotic protein, the Bcl-2 level was not different amongst all groups (Figure 5B). However, the Cyt c levels and cleaved-caspase 3/caspase 3 ratio in all of the Mdivi-1 treated groups were significantly decreased, when compared with the control group (Figure 5C,D, respectively).

Cardiac apoptotic proteins

Figure 5
Cardiac apoptotic proteins

The effect of Mdivi-1 given at different time-points (pretreatment, treated during ischemia, and onset of reperfusion) on cardiac apoptosis. (A) Bax. (B) Bcl-2. (C) Cyt c. (D) Cleaved caspase-3/caspase-3. n=4 per group. *P<0.05 compared with sham; P<0.05 compared with control.

Figure 5
Cardiac apoptotic proteins

The effect of Mdivi-1 given at different time-points (pretreatment, treated during ischemia, and onset of reperfusion) on cardiac apoptosis. (A) Bax. (B) Bcl-2. (C) Cyt c. (D) Cleaved caspase-3/caspase-3. n=4 per group. *P<0.05 compared with sham; P<0.05 compared with control.

Moreover, in cardiac I/R injury (control group), the TUNEL positive cells were significantly increased, compared with the sham-operated group (Figure 6). All Mdivi-1 treated groups showed significantly decreased TUNEL positive cells, compared with the control groups, and the TUNEL positive cells were markedly decreased in a pretreated group, compared with those in the groups receiving Mdivi-1 during-ischemia and upon the onset of reperfusion (Figure 6).

Cardiac myocyte apoptosis using the TUNEL-positive cells

Figure 6
Cardiac myocyte apoptosis using the TUNEL-positive cells

The effect of Mdivi-1 given at different time-points (pretreatment, treated during ischemia and onset of reperfusion) on TUNEL-positive cells. n=4 per group. *P<0.05 compared with sham; P<0.05 compared with control; P<0.05compared with pretreated.

Figure 6
Cardiac myocyte apoptosis using the TUNEL-positive cells

The effect of Mdivi-1 given at different time-points (pretreatment, treated during ischemia and onset of reperfusion) on TUNEL-positive cells. n=4 per group. *P<0.05 compared with sham; P<0.05 compared with control; P<0.05compared with pretreated.

Mdivi-1 attenuated cardiac mitochondrial dysfunction

Cardiac I/R was associated with mitochondrial dysfunction by increasing mitochondrial ROS production, membrane potential depolarization and swelling, when compared with sham operation (Figure 7A–C). However, in all Mdivi-1 treated groups, cardiac mitochondrial ROS levels were significantly decreased, when compared with the control group (Figure 7A). In the case of mitochondrial membrane potential changes (Figure 7B), all Mdivi-1 treated groups had a significantly increased mitochondrial membrane potential, compared with the ischemic control group, indicating decreased mitochondrial depolarization. Consistent with this, mitochondrial swelling was significantly decreased in all Mdivi-1 treated groups, indicated by increased absorbance, compared with the control group (Figure 7C). However, the pretreated group had a significantly higher level than those in the groups receiving Mdivi-1 during and upon the onset of reperfusion (Figure 7C). Representative transmission electron micrographs of cardiac mitochondria showed that mitochondria in the ischemic area of the control group had evidence of the unfolding of cristae, indicating mitochondrial swelling, when compared with sham group (Figure 7D). However, in the Mdivi-1 treated groups, less cardiac mitochondrial swelling was observed (Figure 7D).

Mitochondrial function parameters and TEM images

Figure 7
Mitochondrial function parameters and TEM images

The effect of Mdivi-1 given at different time-points (pretreatment, treated during ischemia, and onset of reperfusion) on cardiac mitochondrial function. (A) Mitochondrial ROS production. (B) Mitochondrial membrane potential. (C) Mitochondrial swelling. (D) TEM representative images of cardiac mitochondria; Scale bar: 600 nm. n=4 per group. *P<0.05 compared with sham; P<0.05 compared with control; P<0.05 compared with pretreated.

Figure 7
Mitochondrial function parameters and TEM images

The effect of Mdivi-1 given at different time-points (pretreatment, treated during ischemia, and onset of reperfusion) on cardiac mitochondrial function. (A) Mitochondrial ROS production. (B) Mitochondrial membrane potential. (C) Mitochondrial swelling. (D) TEM representative images of cardiac mitochondria; Scale bar: 600 nm. n=4 per group. *P<0.05 compared with sham; P<0.05 compared with control; P<0.05 compared with pretreated.

Mdivi-1 improved cardiac mitochondrial dynamics

During cardiac I/R, the mitochondrial Drp1 expression level was significantly increased in cardiac I/R injury group, compared with sham group (Figure 8A). However, all Mdivi-1 treated groups were significantly decreased the Drp1 expression in mitochondria, when compared with the control group (Figure 8A). In the cytosolic fraction, the expression level of phosphorylated Drp1 at Ser616, which occurred during the activation of the mitochondrial fission process, was significantly increased in the control, compared with sham group (Figure 8B). While, all Mdivi-1 treated groups showed a significant decrease in Drp1 (Ser616) phosphorylation level, when compared with the control group (Figure 8B). In contrast with mitochondrial fission proteins, there was no difference in the levels of mitochondrial fusion proteins, including Mfn2 (Figure 8C) and OPA1 (Figure 8D), between the sham, control, and all of the Mdivi-1 treated groups.

Levels of mitochondrial fission and fusion protein expression

Figure 8
Levels of mitochondrial fission and fusion protein expression

The effect of Mdivi-1 given at different time-points (pretreatment, treated during ischemia, and onset of reperfusion) on cardiac mitochondrial fission and fusion. (A) Mitochondrial Drp1 level. (B) Phosphorylated Drp1 at Ser616 in cytosol. (C) Mitochondrial Mfn2 level. (D) Mitochondrial OPA1 level. n=4 per group. Abbreviations: OPA1, pDrp1 (Ser616), phosphorylation of dynamin related protein-1 at Ser616; VDAC, voltage-dependent anion channel. *P<0.05 compared with sham; P<0.05 compared with control.

Figure 8
Levels of mitochondrial fission and fusion protein expression

The effect of Mdivi-1 given at different time-points (pretreatment, treated during ischemia, and onset of reperfusion) on cardiac mitochondrial fission and fusion. (A) Mitochondrial Drp1 level. (B) Phosphorylated Drp1 at Ser616 in cytosol. (C) Mitochondrial Mfn2 level. (D) Mitochondrial OPA1 level. n=4 per group. Abbreviations: OPA1, pDrp1 (Ser616), phosphorylation of dynamin related protein-1 at Ser616; VDAC, voltage-dependent anion channel. *P<0.05 compared with sham; P<0.05 compared with control.

Discussion

Mitochondrial fission inhibition giving after myocardial ischemic insults still exerts cardioprotection

The beneficial effects of inhibiting mitochondrial fission prior to the induction of ischemia in in vitro [8,9,15–18,27], ex vivo [8–10], and in vivo [15,18,19] studies have been reported. However, this application is not clinically relevant since patients with AMI naturally only come to the hospital after they experience symptoms of coronary occlusion. Since the benefits of the mitochondrial fission inhibitor, Mdivi-1, given after myocardial ischemia is not known, we sought to compare the differential temporal effects of Mdivi-1 given at three different time-points including pretreatment, treated during ischemia and also at the onset of reperfusion in rats with cardiac I/R injury. If proved beneficial in cardiac I/R, this intervention could have provided some insights regarding its potential clinical benefits in the near future.

The major findings from the present study clearly demonstrated that cardiac I/R injury caused an increased infarct size and cardiac arrhythmia, LV dysfunction, mitochondrial dysfunction, and also increased mitochondrial fission in the heart. Our results demonstrated for the first time that inhibition of mitochondrial fission with Mdivi-1 during I/R injury still exerted cardioprotective benefits even when it was given after myocardial ischemia had already occurred. These benefits included: (i) attenuation of cardiac mitochondrial dynamic imbalance as indicated by decreased mitochondrial fission; (ii) attenuation of cardiac mitochondrial dysfunction as indicated by a reduction in mitochondrial ROS production, mitochondrial membrane depolarization, and mitochondrial swelling; (iii) a decrease in arrhythmia score and an increase in Cx43 phosphorylation; (iv) a decrease in infarct size, apoptotic cells (TUNEL positive cells), and pro-apoptotic protein (Caspase 3, Bax, and Cyt c) expression. All of these effects instigated by the administration of Mdivi-1 could be responsible for (v) the improvement in LV function as demonstrated in the present study.

Mdivi-1 given after myocardial ischemia effectively reduced the infarct size, improved cardiac function, and reduced mortality rate

All cardiac I/R injury studies have been mainly focusing on a reduction in infarct size [2,3,28]. Although previous reports had shown the beneficial effects of Mdivi-1 pretreatment during I/R injury by reducing myocardial infarct size [9,15,29], its effects given after cardiac ischemia were not known. Our study demonstrated that Mdivi-1 treatment could still provide cardioprotective effects even when it was given after ischemia since all Mdivi-1-treated rats had markedly reduced infarct size. Moreover, a previous study by Sharp et al. [11] demonstrated that Mdivi-1 administration during cardiopulmonary resuscitation (CRP) in rat models of KCl-induced cardiac arrest provided benefits to the heart such as improved cardiac function and mitochondrial function. However, the KCl-induced cardiac arrest caused global ischemia, which is not often observed in the clinical setting of AMI. In contrast, LAD ligation-induced regional ischemia was used as a model of AMI in the present study, which usually occurs in patients with AMI.

The present study also demonstrated for the first time the comparative effects of Mdivi-1 given at different time-points during I/R injury. Interestingly, we found that Mdivi-1 treatment during ischemia and upon the onset of reperfusion also led to attenuated myocardial infarct size (56 and 53% reduction for the treatment during-ischemia and at the onset of reperfusion, respectively). However, the greatest effect was seen on infarct size reduction in the pretreated group (~83% reduction). The underlying mechanism for the reduction in the infarct size by Mdivi-1 could be due to its ability to attenuate cardiac mitochondrial dysfunction and mitochondrial fission and decrease the apoptotic cells. Taken together, all of the aforementioned benefits of Mdivi-1 treatment could be responsible for improved LV function as observed in the present study.

Inhibition of mitochondrial fission attenuated cardiac mitochondrial dysfunction and apoptosis, and improved mitochondrial dynamics during cardiac I/R injury

It is known that cardiac I/R injury is associated with mitochondrial damage and cardiac cell apoptosis [8,9,15,17]. The mitochondrial damage was initiated after 20 min of ischemia [30]. Moreover, in a model of 45-min occlusion followed by reperfusion for either 30 min, 1, 3, 6, 12, or 24 h it was shown that mitochondrial damage gradually increased after reperfusion with the maximum reach of the damage after 3 h of reperfusion [31]. Therefore, mitochondrial damage can occur after 20 min of occlusion and the severity will be gradually increased during reperfusion period. Furthermore, our results clearly demonstrated an increase in Drp1 activation and translocation to the mitochondria during I/R as indicated by increased phosphorylation of Drp1 in the cytosolic fraction along with increased mitochondrial Drp1, thus impairing the balance of mitochondrial dynamics. According to the pharmacokinetics of Mdivi-1, Cui et al. (2016) [17] is the only report studying in vivo pharmacokinetic profile of Mdivi-1 conducted till date. They found that intraperitoneal injection of Mdivi-1 at 20 mg/kg resulted in peak plasma and brain concentrations 2 and 4 h later, respectively, with a half-life estimated to be 12 h [17]. Since the plasma half-life of Mdivi-1 is long, Mdivi-1 given during ischemia or at the onset of reperfusion could improve mitochondrial dynamics and cardiac function following I/R injury. It is also possible that the drug might reach the infarcted zone upon onset of reperfusion, and therefore might explain why there is no substantial difference between treatment during ischemia and that after reperfusion. Moreover, mitochondria undergoing the fission process, concomitantly with Bax activation, could lead to increased Bax-induced Cyt c release from mitochondria, thus promoting caspase activation and cardiomyocyte apoptosis [32,33]. This could explain our findings that all Mdivi-1 treated groups had significantly decreased Drp1 expression, which resulted in reduced Cyt c, Cleaved-caspase 3, and cardiac cells apoptosis.

In addition, the impairment in cardiac mitochondrial function including increased cardiac mitochondrial ROS production, mitochondrial membrane depolarization, and mitochondrial swelling caused by I/R injury were significantly reduced in all Mdivi-1 treated groups. Despite the levels of caspase 3 activation, Drp1 expression and phosphorylation were not different amongst three time-points of treatment, cardiac mitochondrial swelling was significantly improved in the pretreatment group, compared with the other groups. Our findings clearly demonstrated that mitochondrial swelling might be responsible for the greater attenuation in infarct size and mortality rate in the pretreatment group. Although this benefit is not applicable in a real clinical setting, the fact that inhibition of mitochondrial fission by Mdivi-1 given during ischemia or reperfusion still exerted cardioprotection provided a significant insight for potential clinical investigation in the future.

Inhibition of excessive mitochondrial fission showed an anti-arrhythmic effect during cardiac I/R injury

In addition to the decreased infarct size and improved LV function, our results also clearly demonstrated that Mdivi-1 pretreatment had a lower arrhythmia score than the control group. Therefore, Mdivi-1 exerts an anti-arrhythmic effect on the heart during the period of I/R. Since oscillation of the mitochondrial membrane facilitated fatal cardiac arrhythmia [34], one possible mechanism could be through its ability to reduce cardiac mitochondrial depolarization caused by I/R injury. In addition, the anti-arrhythmic effects of Mdivi-1 could be due to its effect on Cx43. Cx43 is a cardiac gap junction protein, which facilitates cardiomyocyte communication via the electrical current flow [35]. Phosphorylation of Cx43 at Ser368 increased the trafficking of Cx43 to the plasma membrane, leading to the generation of a gap junction [35]. In this study, pCx43 was decreased in cardiac I/R, whereas it was increased in the Mdivi-1 treated groups. All of these effects of Mdivi-1 together with infarct size reduction could be responsible for the reduction in arrhythmia found in this study.

In conclusion, although mitochondrial fission is essential for proper cardiac function under physiologic condition [11,15], excessive mitochondrial fission due to cardiac I/R injury needs to be prevented. As a result, inhibition of excessive mitochondrial fission due to I/R injury was shown to be cardioprotection. During cardiac I/R injury, excessive mitochondrial fission leads to increased mitochondrial dysfunction and finally cardiac dysfunction. The drug Mdivi-1 effectively reduces excessive mitochondrial fission and mitophagy to the proper level, thus attenuating stressful mitochondrial status. The present study is also the first in vivo investigation to demonstrate the cardioprotective benefits of mitochondrial fission inhibitor Mdivi-1 given even after myocardial ischemia and during reperfusion, thus providing significant insights for future clinical investigations.

Clinical perspectives

  • Patients with AMI naturally only receive the reperfusion therapy when experience symptoms of coronary occlusion, to attenuate cardiac I/R injury, the effective drugs given during ischemic period or upon the onset of reperfusion are more clinically relevant than treatment prior to myocardial ischemia.

  • Mdivi-1 administered prior to ischemia, during-ischemia, and upon the onset of reperfusion improved cardiac mitochondrial function and cardiac function during I/R injury.

  • Mitochondrial fission inhibitor given after myocardial ischemia effectively improved cardiac function encourages further clinical study to warrant its clinical usefulness in AMI patients.

Competing interests

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

Funding

This work was supported by the Thailand Research Fund grants TRF-Royal Golden Jubilee Program [grant numbers PHD/0144/2558 (to C.M. and NC), RTA6080003 (to S.C.C.), RSA6180056 (to S.P.)]; the NSTDA Research Chair grant from the National Science and Technology Development Agency Thailand (to N.C.)]; and the Chiang Mai University Center of Excellence Award.

Author contribution

S.C.C. and N.C. conceived and designed the experiments. C.M. and S.P. conducted the experiments. C.M., S.P., S.K., T.J., S.C.C., and N.C. analyzed the data. All authors reviewed the manuscript.

Abbreviations

     
  • AAR

    area at risk

  •  
  • AMI

    acute myocardial infarction

  •  
  • Cx43

    Connexin-43

  •  
  • Cyt c

    cytochrome c

  •  
  • DCF

    dichlorofluorescein

  •  
  • DCFDA

    dichlorohydro-fluorescein diacetate dye

  •  
  • Drp1

    dynamin-related protein 1

  •  
  • ECG

    electrocardiogram

  •  
  • HR

    heart rate

  •  
  • I

    ischemia

  •  
  • I/R

    ischemia/reperfusion

  •  
  • JC-1

    5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide dye

  •  
  • LAD

    left anterior descending

  •  
  • LV

    left ventricular

  •  
  • LVEDP

    LV end-diastolic pressure

  •  
  • LVEF

    LV ejection fraction

  •  
  • LVESP

    LV end-systolic pressure

  •  
  • Mdivi-1

    mitochondrial division inhibitor 1

  •  
  • Mfn2

    mitofusin 2

  •  
  • OPA1

    optic atrophy 1

  •  
  • P-V loop

    pressure-volume loop

  •  
  • R

    reperfusion

  •  
  • ROS

    reactive oxygen species

  •  
  • SV

    stroke volume

  •  
  • TTC

    2,3,5-triphenyltetrazolium chloride

  •  
  • TUNEL

    terminal deoxynucleotidyl transferase dUTP nick-end labeling

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