Ischemia reperfusion (I/R) injury is a common event following myocardial infarction (MI) resulting in excessive oxidative stress, calcium overload, inflammation, and cardiomyocyte death. Mitochondrial homeostasis including their dynamics are imbalanced in cardiac I/R injury in favor of increased mitochondrial fission. Inhibition of mitochondrial fission prior to I/R injury is protective and improves cardiac function following MI. Clinically, patients with MI often receive treatment following initiation of the ischemic event. Thus, treatments with more realistic timing would have better translational value and are important to research. In a recent study published in Clinical Science, Maneechote et al. [Clin. Sci. (2018) 132, 1669–1683] examined the effect of inhibiting mitochondrial fission using the mitochondrial division inhibitor (Mdivi-1) at different time points, pre-ischemia, during-ischemia, and upon onset of reperfusion, in a rat cardiac I/R model. The findings showed the greatest cardiac function improvement with pre-ischemia treatment along with decreased mitochondrial fragmentation and increased mitochondrial function. Mdivi-1 given during ischemia and at onset of reperfusion also improved cardiac function, but to a lesser extent than pre-ischemia intervention. Maneechote et al. postulated that the LV protection by Mdivi-1 in cardiac I/R could be due to an improvement in mitochondrial dysfunction through attenuating excessive mitochondrial fission which then reduces apoptotic myocytes. Their findings provide new insights into future treatment of patients suffering acute MI which could consider targetting the excessive mitochondrial fission during cardiac ischemia or at onset of reperfusion. Here, we will further discuss the background of the study, potential molecular mechanisms of mitochondrial fission, consequences of the fission, and future research directions.

Acute myocardial infarction (MI) is the leading cause of death worldwide [1]. This event involves the occlusion of a coronary artery preventing sufficient blood flow to an area of the heart and causing cardiac ischemia and subsequent loss of cardiac myocytes. The standard clinical treatment is restoration of blood flow by myocardial reperfusion therapy [2]. However, ischemia/reperfusion injury (I/R) is a prevalent sequela of the reperfusion therapy. Extensive research has been done to identify the events leading to cardiac damage and potential interventions that could provide cardioprotection. A number of mechanisms such as I/R induced excessive reactive oxygen species (ROS) production, intracellular calcium overload, cardiac contractile dysfunction, apoptotic, and necrotic cell death are all thought to contribute to cardiac dysfunction after cardiac I/R [3]. Moreover, following I/R injury mitochondria appear less efficient, produce more ROS, and induce apoptosis through activation of intrinsic apoptotic pathways, cytochrome c release, and caspase activation. Contributing to this series of events is the opening of the mitochondrial permeability transition pore (mPTP) due to mitochondrial calcium overload, ATP depletion, oxidative stress, and loss of mitochondrial matrix pH [3].

A new research direction involves the identified role of mitochondrial dynamics in cardiac I/R injury. Mitochondrial dynamics, the process including fission and fusion of mitochondria, is crucial for mitochondrial homeostasis. Several nuclear-encoded GTPase proteins mediate both of these processes. During mitochondrial fusion, mitofusin 1 and 2 (Mfn1/2) form hetero-oligomers to mediate outer mitochondrial membrane fusion and optic atrophy protein 1 (Opa1), in its long isoform, facilitates inner mitochondrial membrane fusion [4]. A GTPase, dynamin-related protein 1 (Drp1), is the master regulator of mitochondria fission. Upon activation, Drp1 translocates from the cytosol to the mitochondrial outer membrane where it binds various adaptor proteins. Drp1 then forms an oligomer ring around the mitochondrion, allowing for the scission event. Thus, Drp1 is believed to facilitate removal of damaged mitochondria by mitophagy and the concurrent biogenesis of new mitochondria thereby promoting a functional pool [5]. Asymmetric Drp1-mediated mitochondrial fission allows for sequestering of damaged mitochondrial components and their selective removal by mitophagy, while fusion is selective for polarized active mitochondria to protect from mitophagy [6]. Evidence for a relationship between Drp1-mediated mitochondrial fission and biogenesis comes from studies which have manipulated mitochondrial dynamics machinery, both fission and fusion, and saw decreases in the peroxisome proliferator-activated receptor γ coactivator 1α (PGC1-α) gene expression program, a known driver of mitochondrial biogenesis. PGC1-α also facilitates mitochondrial fission and mitophagy suggesting a cross-regulatory circuit exists [7]. In addition, the sirtuins, an evolutionarily conserved family of proteins with NAD-dependent deacetylase activity, are associated with mitochondrial homeostasis and longevity. Specifically, reduced expression of SIRT3, the mitochondria-specific sirtuin, has been implicated in mitochondrial loss induced by angiotensin II in aged mice [8]. Reduced expression of SIRT3 is also observed with renal mitochondrial damage in a mouse model of acute kidney injury, whereas SIRT3 overexpression protects against mitochondrial fission, depolarization, and mitophagy-mediated by Drp1 in a cell culture model of renal injury [9]. Accordingly, it is not surprising that mitochondrial dysfunction and cardiac damage induced by I/R are exaggerated in SIRT3−/− mice [10] and transient overexpression of PGC1-α thus is protective against cardiac I/R [11].

Numerous studies demonstrate cardiac dysfunction in I/R injury is associated with enhanced mitochondrial fission. During I/R, mitophagy seems insufficient to remove damaged mitochondria resulting in excessive mitochondrial fragmentation and dysfunctional mitochondria [12]. Given that calcium is a known inducer of mitochondrial fission, the calcium overload following I/R likely contributes to the excessive mitochondrial fission via calcineurin-mediated dephosphorylation Drp1 at the Ser637 residue [13]. In a rat model of I/R as short as 20-min occlusion followed by 25-min reperfusion causes mitochondrial damage [14,15]. While there are limited studies to genetically target Drp1 in cardiac I/R models, in vitro genetic overexpression of dominant-negative Drp1 in cardiac myocytes in I/R injury models is demonstrated to attenuate mitochondrial fission and dysfunction, thus preventing cell death [16]. Adenoviral transduction of dominant-negative Drp1 via cardiac injection in a rat model of I/R in vivo has also demonstrated to preserve mitochondrial function, decrease infarct size, attenuate cardiomyocyte death, and improve cardiac function [17]. Moreover, pharmacologic inhibition of mitochondrial fission by a Drp1 inhibitor, mitochondrial division inhibitor 1 (Mdivi-1), decreases excessive fission during I/R, inhibited mPTP opening, prevented cardiac myocyte death, and decreased infarct size [16]. In another study looking at cardiac I/R, pharmacologic inhibition of Drp1 by the small molecule inhibitor, P110, decreased infarct size, improved mitochondrial oxygen consumption, and decreased mitochondrial ROS production [18]. To date, the majority of studies have given mitochondrial fission inhibitors including Mdivi-1 prior to the I/R injury demonstrating their cardioprotection [16,18–20]. However, the translational values of these pretreatment studies remain unclear since the patients with acute MI mostly receive medical treatment following the initiation of cardiac ischemia.

In a recent issue of Clinical Science, Maneechote et al. [21] provide the first evidence that Mdivi-1 administered at any point prior, during ischemia or upon the onset of reperfusion improved mitochondrial function and demonstrated cardiac protection in a rat model of cardiac I/R injury. Mdivi-1 given after myocardial ischemia effectively reduced infarct size and mortality rate compared with the control. However, the protective effects were greatest when Mdivi-1 was administered prior to ischemia. Interestingly, the arrhythmia score was only decreased in the Mdivi-1 pretreated group. Cardiac function was assessed as systolic volume, left ventricular end diastolic volume and pressure, dP/dt, and %left ventricular ejection fraction/LVEF. All parameters were improved with Mdivi-1 treatment at any time-point of I/R injury, while the greatest improvement in LVEF was in the pretreatment group. Mdivi-1 treatment also attenuated cellular apoptosis by terminal dUTP nick-end labeling (TUNEL) analysis in all Mdivi-1 treated groups. Again, the pretreated group had the fewest TUNEL positive cells with I/R injury. When isolated mitochondrial function was assessed, Mdivi-1 treatment attenuated all measures of dysfunction increased in I/R when compared with sham-operated rats. These included mitochondrial swelling, membrane potential, and mitochondrial ROS production by cell permeant dichlorofluorescin diacetate staining. Consistent with previous studies, cardiac I/R alone increased mitochondrial fission as measured by total Drp1 expression and expression of Ser616 p-Drp1 in the cytosolic fraction in the control compared with sham groups. Mdivi-1 treatment at any time point of I/R decreased expression levels of Drp1 and p-Drp1 suggesting that the drug treatment decreased activation of mitochondrial fission. A previous pharmacokinetic study of Mdivi-1 suggests that following intraperitoneal injection, it reaches peak levels in plasma and brain at 2 and 4 h, respectively, and has a half-life of approximately 12 h [22]. The authors speculate that this long half-life is what allows for Mdivi-1 to be protective during ischemia or at onset of reperfusion.

Despite finding that pretreatment with Mdivi-1 is most effective at protecting cardiac function with I/R, this manuscript presents novel information that inhibiting mitochondrial fission during cardiac ischemia or the onset of reperfusion still has a significant cardioprotective benefit. These findings are thus clinically relevant to patients experiencing acute MI who seek medical attention once the ischemic attack has initiated. The resounding data that inhibition of mitochondrial fission prior to the ischemic event is most protective suggests that ischemia itself is a significant contributor in the enhanced mitochondrial fission than the reperfusion event. In addition, the differences in studies which have focussed on the ischemic condition and seen excessive mitochondrial fission [23] and those which have looked at the I/R event as initiating mitochondrial fission [16,19] may need to be reconciled to better tease out the mechanism for the mitochondrial fission associated with the onset of cardiac I/R.

Limitations of the study include minimal exploration into the role of mitochondrial fusion and the reliance on the drug Mdivi-1, which may not be a specific Drp1 inhibitor [24]. In terms of mitochondrial fusion, reports on its role in cardiac I/R are inconsistent. Some studies demonstrated that overexpression of mitochondrial fusion proteins such as Mfn1/2 was protective against I/R injury in terms of attenuated cardiomyocyte death and mitochondrial dysfunction [16]. Inhibition of other fusion mediating protein Opa1 with Opa1+/− mice thus enhanced I/R injury [25]. In contrast, a study in which Mfn1/2 were silenced in hearts also saw protection against cardiac I/R injury [26]. In their Clinical Science study, Maneechote et al. examined levels of mitochondrial fusion proteins Mfn2 and Opa1 amongst sham, I/R, and Mdivi-1-treated I/R groups and found no differences. Further investigation into the contribution of mitochondrial fusion in this process would help in understanding whether the increased mitochondrial fission observed with I/R is entirely maladaptive or to some extent, serving as a compensatory mechanism in the pathophysiological environment. At last, finding more selective fission inhibitors and/or ischemic lesion specific delivery may be even more protective and would reduce the risk for off-target effects. Maneechote et al. postulated that the LV protection by Mdivi-1 in cardiac I/R could be due to an improvement in mitochondrial dysfunction through attenuating excessive mitochondrial fission thereby reducing apoptotic myocytes. A schematic diagram of the potential mechanism for cardiac protection by Mdivi-1 is depicted in the Figure 1. Taken together, the above findings from this study provide new insights into future treatment for the patient with acute MI which could consider targetting the excessive mitochondrial fission during cardiac ischemia or at onset of reperfusion.

Schematic diagram of cardiac I/R injury mechanism of excessive mitochondrial fission leading to increased apoptosis and cardiac dysfunction.

Figure 1
Schematic diagram of cardiac I/R injury mechanism of excessive mitochondrial fission leading to increased apoptosis and cardiac dysfunction.

Pharmacological inhibition of Drp1 driven mitochondrial fission by Mdivi-1 prior to ischemia, during ischemia or at onset of reperfusion all provided cardiac protection compared to control. ROS, reactive oxygen species; mPTP, mitochondrial permeability transition pore.

Figure 1
Schematic diagram of cardiac I/R injury mechanism of excessive mitochondrial fission leading to increased apoptosis and cardiac dysfunction.

Pharmacological inhibition of Drp1 driven mitochondrial fission by Mdivi-1 prior to ischemia, during ischemia or at onset of reperfusion all provided cardiac protection compared to control. ROS, reactive oxygen species; mPTP, mitochondrial permeability transition pore.

Funding

This work was supported by the National Institute of Health [grant numbers HL128324, HL133248, DK111042 (to S.E.)]; and the American Heart Association [grant number 16GRNT30410007 (to S.E.)].

Competing interests

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

Abbreviations

     
  • Drp1

    dynamin-related protein 1

  •  
  • I/R

    ischemia/reperfusion

  •  
  • LV

    left ventricle

  •  
  • Mdivi-1

    mitochondrial division inhibitor

  •  
  • Mfn1/2

    mitofusin 1 and 2

  •  
  • MI

    myocardial infarction

  •  
  • mPTP

    mitochondrial permeability transition pore

  •  
  • Opa1

    optic atrophy protein 1

  •  
  • PGC1-α

    proliferator-activated receptor γ coactivator 1α

  •  
  • SIRT3

    sirtuin 3

  •  
  • ROS

    reactive oxygen species

  •  
  • TUNEL

    terminal dUTP nick-end labeling

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