Abstract

Mitochondria are dynamic, undergoing both fission and fusion. Evidence indicates that a balance between these two processes is necessary to maintain a healthy state. With ischemia/reperfusion (I/R) of the heart, fission is enhanced and is associated with mitochondrial swelling, depolarization, and production of reactive oxygen species (ROS), as well as apoptosis. Blocking fission is effective in reducing I/R-induced tissue damage and contractile dysfunction. In a groundbreaking study appearing in Clinical Science, Maneechote et al. assessed whether correcting the imbalance in mitochondrial dynamics with I/R by enhancing fusion would also be protective. Using a rat model, they investigated the efficacy of pharmacological intervention with mitochondrial fusion promoter-M1 (M1) given before ischemia, during ischemia, or at the onset of reperfusion. With pretreatment being the most effective, they found that M1 attenuated the incidence of arrhythmias, reduced infarct size, preserved cardiac function, and decreased mortality. M1 reduced I/R-induced increases in cytosolic cytochrome c, cleaved caspase 3, and apoptosis. All M1 groups exhibited modestly attenuated I/R-induced mitochondrial ROS levels and swelling, and preserved mitochondrial membrane potential. M1 also prevented a decrease in complex V levels with I/R. However, exactly how M1 stimulates mitochondrial fusion is unclear and other nonfusion-related actions of this phenylhydrazone compound should be considered, such as anti-oxidant actions, preconditioning signaling, or effects on putative mitochondrial connexin 43.

Heart attacks are a leading cause of death worldwide with no effective pharmacological strategies to limit permanent damage to the heart. However, accumulating preclinical evidence has focused attention on targeting mitochondria, which occupy about a third of the volume of an adult cardiomyocyte and are a major source of damaging reactive oxygen species (ROS) with reperfusion. One approach involves tempering complex I activity so as to limit formation of ROS, for instance with metformin [1]. An alternative strategy is built around the fact that mitochondria are dynamic, undergoing fission or fusion, which is necessary for both the cell and mitochondria to maintain a healthy state. Normally a balance occurs between fission and fusion, such that an increase in one over the other has harmful consequences. Indeed, in a seminal study, Song et al. [2] demonstrated that a lack of mitochondrial dynamism in cardiac myocytes is less deleterious than dynamic imbalance. Surprisingly, under nonstressed conditions, mitochondrial size (fragmentation or enlargement) would seem to be a less important determinant of cardiac phenotype or pathology than the impact that dynamic imbalance has via dysregulated mitochondria quality control, as evidenced by alterations in mitophagy or mitogenesis.

The GTPase dynamin-related protein 1 (Drp1) plays a critical role in mitochondrial fission (Figure 1). In response to certain stimuli, cytosolic Drp1 translocates to mitochondria, where it oligomerizes at the future fission site and forms a ring. The GTP-dependent constriction of this ring leads to mitochondrial division. Mitochondrial fission factor (Mff), an outer mitochondrial membrane protein, is also important in fission by binding Drp1. Several studies have documented increased mitochondrial fission in isolated cardiac myocytes of whole hearts with ischemia or reperfusion (I/R) [3–7]. Additionally, knockdown of Drp1 or its pharmacological blockade with mitochondrial division inhibitor 1 (mdivi-1) has been shown to have remarkable protective effects on cardiac myocyte viability and heart function. Besides the anticipated changes in mitochondrial size, diminishing the actions of Drp1 during I/R is associated with reduction in infarct size, diastolic dysfunction, incidence of arrhythmias, ROS formation, cytochrome c release, caspase 3 activation, and apoptosis. Targeting Drp1 is most effective if done prior to ischemia, but still effective if done during ischemia; however, conflicting results are reported for the effectiveness of targeting Drp1 during re-oxygenation [3,4]. Attenuating mitochondrial fission by targeting Drp1 may have relevance in sudden cardiac arrest as well [8]. Although not proven, the simplest explanation for the beneficial consequences of blocking fission is that, compared with elongated mitochondria, fragmented mitochondria are more susceptible to swelling and outer membrane permeabilization, and less able to accommodate a calcium load and oxidative stress before undergoing mitochondrial permeability transition pore (mPTP) opening. Elongated mitochondria may have a higher respiratory capacity as well.

Mitochondrial dynamics and ischemia/reperfusion (I/R) injury

Figure 1
Mitochondrial dynamics and ischemia/reperfusion (I/R) injury

With I/R, Drp1 translocates to mitochondria, where it oligomerizes and participates in the fission process. Fragmented mitochondria are more susceptible to swelling, mPTP (mitochondrial permeability transition pore) opening, and ROS production. Bax is recruited as well, enhancing mitochondrial outer membrane permeabilization, cytochrome c release, caspase activation, and apoptosis. Inhibiting Drp1 with mdivi-1 (mitochondrial division inhibitor 1) reduces I/R injury to the heart. The utility of the alternative approach of enhancing fusion was assessed by Maneechote et al. using mitochondrial fusion promoter-M1 or hydrazone M1 (M1), which may act in part by increasing expression levels of complex V subunits (ATP5A/B). M1 was found to limit I/R injury and preserve cardiac function. M1 attenuated I/R-induced mitochondrial ROS and prevented mitochondrial depolarization, which were associated with reduced cytochrome c release, caspase 3 cleavage, and apoptosis. Nonfusion-related actions of M1, for instance via a mitochondrial pool of Cx43 and reduced reverse electron transfer, cannot be discounted. See text for additional details.

Figure 1
Mitochondrial dynamics and ischemia/reperfusion (I/R) injury

With I/R, Drp1 translocates to mitochondria, where it oligomerizes and participates in the fission process. Fragmented mitochondria are more susceptible to swelling, mPTP (mitochondrial permeability transition pore) opening, and ROS production. Bax is recruited as well, enhancing mitochondrial outer membrane permeabilization, cytochrome c release, caspase activation, and apoptosis. Inhibiting Drp1 with mdivi-1 (mitochondrial division inhibitor 1) reduces I/R injury to the heart. The utility of the alternative approach of enhancing fusion was assessed by Maneechote et al. using mitochondrial fusion promoter-M1 or hydrazone M1 (M1), which may act in part by increasing expression levels of complex V subunits (ATP5A/B). M1 was found to limit I/R injury and preserve cardiac function. M1 attenuated I/R-induced mitochondrial ROS and prevented mitochondrial depolarization, which were associated with reduced cytochrome c release, caspase 3 cleavage, and apoptosis. Nonfusion-related actions of M1, for instance via a mitochondrial pool of Cx43 and reduced reverse electron transfer, cannot be discounted. See text for additional details.

Fission accomplishment may also be dependent upon the final level of the pro-apoptotic protein Bax that is recruited to the mitochondrial outer membrane, which in turn may be determined by Drp1 [9]. During I/R, Bax translocates to the mitochondria and controls permeability of the mitochondrial outer membrane and cytochrome c release. In cardiac myocytes, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) may promote I/R injury by suppressing Bax inhibitor-1 (BI-1) activity [10].

The role of mitochondrial fusion with I/R is unresolved, the key proteins being the outer membrane proteins and GTPases, mitofusin 1 and 2 (Mfn1 and Mfn2), and the inner membrane protein, optic atrophy 1 (OPA1). Reduced OPA1 protein levels are associated with ischemia [11], and Mfn2 activity is linked to suppression of Bax activation and free radical-induced mitochondrial depolarization [12]. But implicating these proteins in I/R injury is complicated by their extra-fusion functions; for instance, hearts deficient in both Mfn1 and Mfn2 are protected against acute infarction due to impaired mitochondria/SR tethering [13].

It is against this background, that Maneechote et al. [14] addressed the provocative question of whether enhancing fusion would protect the heart from I/R injury (Figure 1). Using a rat model of 30-min ischemia by coronary occlusion followed by 120-min reperfusion, they investigated the efficacy of pharmacological intervention with the mitochondrial fusion promoter-M1 or hydrazone M1 (M1) given at various times: 15 min before ischemia, during ischemia, and with the onset of reperfusion. With pretreatment being the most effective, they found that M1 attenuated the incidence of arrhythmias and reduced infarct size, which was associated with preserved cardiac function and decreased mortality. M1 suppressed apoptosis as indexed by a TUNEL assay at the end of reperfusion. This was accompanied by decreased protein levels of Bax (relative to VDAC, but whether these levels are mitochondrial is unclear), cytosolic (presumably) cytochrome c, and cleaved caspase 3. Pretreatment before ischemia was the most effective strategy. Notably, M1 had only modest effects in suppressing I/R-induced increases in mitochondrial levels of Drp1 (total and the active phosphorylated form), although it did preserve or slightly enhance mitochondrial levels of the fusion proteins Mfn2 and OPA1. M1 prevented a modest decrease in complex V levels with I/R, but there was no significant effect of MI on the levels of the other respiratory complexes. All M1 groups modestly attenuated I/R-induced mitochondrial ROS levels and mostly prevented mitochondrial depolarization. Mitochondrial swelling with I/R was decreased in all M1 treated groups, as measured spectrophotometrically and by less unfolding of mitochondrial cristae in transmission electron micrographs. Notably absent are electron micrographic images of sections of the heart showing fragmented mitochondria with I/R and elongated mitochondria with M1 treatment.

Details on how M1 promotes mitochondrial fusion are scant. This cell-permeable phenylhydrazone compound was identified as the most active among 75,000 in rescuing fusion in Mfn1 or Mfn2 knockout mouse embryonic fibroblasts (MEFs) [15]. In that study, M1 was found to increase protein levels of the two major components (ATP5A and ATP5B) of the catalytic center of ATP synthase (mitochondrial complex V) by undefined means, an action consistent with the preservation of complex V levels in the study by Maneechote et al. [14]. However, the role of complex V in mitochondrial dynamism is not established; plus, it is difficult to attribute the actions of M1 observed by Maneechote et al. to increased ATP5A and ATP5B protein levels (which were not measured) given the timeframe involved.

Alternative explanations for the cardioprotective effects of M1 cannot be dismissed. A potential direct or indirect antioxidant action of M1 might be considered [16]. The finding of Maneechote et al. that M1 acted to maintain Ser368 phosphorylation of connexin 43 (Cx43) suggests that M1 has effects on signaling events in cardiac myocytes. This action of M1 was effective with pretreatment, but did show a similar trend with treatment during ischemia or reperfusion. The finding was presented in the context of arrhythmias, as sarcolemmal Cx43 is a constituent of gap junction channels and contributor to reperfusion arrhythmias. Ser368 phosphorylation would limit channel activity and intercellular communication. But a pool of Cx43 is reported as well in subsarcolemmal mitochondria, which are most susceptible to I/R injury, and is linked to preconditioning [17]. Moreover, reduced mitochondrial Cx43 function may dampen complex I activation or the reverse electron transfer to complex I and ROS production during reperfusion [17]. Most of the mitochondrial Cx43 appears to be phosphorylated, with Ser368 phosphorylation linked to cardioprotection [18,19].

In conclusion, Maneechote et al. [14] are to be commended for an innovative study that assesses the potential therapeutic strategy of enhancing mitochondrial fusion to protect the heart from I/R injury. Further study is required to establish the long-term consequences of M1 treatment on heart structure and function, as well as mortality. Importantly, the molecular basis by which M1 is acting must be clearly determined. In this regard, additional evidence is needed to establish the relative importance of mitochondrial fusion versus putative non-fusion based actions of M1 in cardioprotection. Nevertheless, this is the first in vivo study to demonstrate the therapeutic benefit to the heart of M1, in this case, to protect the heart from I/R injury. A notable feature of the study is that treatment was effective when given during the ischemic period or at the onset of reperfusion, although treatment prior to myocardial ischemia was the most effective. Consequently, these results have broad clinical implications and may herald the development of an entirely new class of cardioprotective drugs.

Competing Interests

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

Funding

This work was supported by a grant to F.A.Z. from the American University of Beirut Faculty of Medicine (MPP – 320145). G.W.B. acknowledges the support of the Department of Pharmacology and Toxicology (UMMC).

Abbreviations

     
  • Cx43

    connexin 43

  •  
  • Drp1

    dynamin-related protein 1

  •  
  • I/R

    ischemia/reperfusion

  •  
  • mdivi-1

    mitochondrial division inhibitor 1

  •  
  • MEF

    mouse embryonic fibroblast

  •  
  • Mff

    mitochondrial fission factor

  •  
  • mPTP

    mitochondrial permeability transition pore

  •  
  • ROS

    reactive oxygen species

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