Mitochondria are no longer considered to be solely the static powerhouses of the cell. While they are undoubtedly essential to sustaining life and meeting the energy requirements of the cell through oxidative phosphorylation, they are now regarded as highly dynamic organelles with multiple functions, playing key roles in cell survival and death. In this review, we discuss the emerging role of mitochondrial fusion and fission proteins, as novel therapeutic targets for treating a wide range of cardiovascular diseases.LINKED ARTICLESThis article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8

Mitochondria alter their shape by undergoing cycles of fusion and fission. Changes in mitochondrial morphology impact on the cellular response to stress, and their interactions with other organelles such as the sarcoplasmic reticulum (SR). Inhibiting mitochondrial fission can protect the heart against acute ischemia/reperfusion (I/R) injury. However, the role of the mitochondrial fusion proteins, Mfn1 and Mfn2, in the response of the adult heart to acute I/R injury is not clear, and is investigated in this study. To determine the effect of combined Mfn1/Mfn2 ablation on the susceptibility to acute myocardial I/R injury, cardiac-specific ablation of both Mfn1 and Mfn2 (DKO) was initiated in mice aged 4–6 weeks, leading to knockout of both these proteins in 8–10-week-old animals. This resulted in fragmented mitochondria (electron microscopy), decreased mitochondrial respiratory function (respirometry), and impaired myocardial contractile function (echocardiography). In DKO mice subjected to in vivo regional myocardial ischemia (30 min) followed by 24 h reperfusion, myocardial infarct size (IS, expressed as a % of the area-at-risk) was reduced by 46% compared with wild-type (WT) hearts. In addition, mitochondria from DKO animals had decreased MPTP opening susceptibility (assessed by Ca2+-induced mitochondrial swelling), compared with WT hearts. Mfn2 is a key mediator of mitochondrial/SR tethering, and accordingly, the loss of Mfn2 in DKO hearts reduced the number of interactions measured between these organelles (quantified by proximal ligation assay), attenuated mitochondrial calcium overload (Rhod2 confocal microscopy), and decreased reactive oxygen species production (DCF confocal microscopy) in response to acute I/R injury. No differences in isolated mitochondrial ROS emissions (Amplex Red) were detected in response to Ca2+ and Antimycin A, further implicating disruption of mitochondria/SR tethering as the protective mechanism. In summary, despite apparent mitochondrial dysfunction, hearts deficient in both Mfn1 and Mfn2 are protected against acute myocardial infarction due to impaired mitochondria/SR tethering.

Novel therapeutic targets are required to protect the heart against cell death from acute ischemia–reperfusion injury (IRI). Mutations in the DJ-1 (PARK7) gene in dopaminergic neurons induce mitochondrial dysfunction and a genetic form of Parkinson's disease. Genetic ablation of DJ-1 renders the brain more susceptible to cell death following ischemia–reperfusion in a model of stroke. Although DJ-1 is present in the heart, its role there is currently unclear. We sought to investigate whether mitochondrial DJ-1 may protect the heart against cell death from acute IRI by preventing mitochondrial dysfunction. Overexpression of DJ-1 in HL-1 cardiac cells conferred the following beneficial effects: reduced cell death following simulated IRI (30.4±4.7% with DJ-1 versus 52.9±4.7% in control; n=5, P<0.05); delayed mitochondrial permeability transition pore (MPTP) opening (a critical mediator of cell death) (260±33 s with DJ-1 versus 121±12 s in control; n=6, P<0.05); and induction of mitochondrial elongation (81.3±2.5% with DJ-1 versus 62.0±2.8% in control; n=6 cells, P<0.05). These beneficial effects of DJ-1 were absent in cells expressing the non-functional DJ-1L166P and DJ-1Cys106A mutants. Adult mice devoid of DJ-1 (KO) were found to be more susceptible to cell death from in vivo IRI with larger myocardial infarct sizes (50.9±3.5% DJ-1 KO versus 41.1±2.5% in DJ-1 WT; n≥7, P<0.05) and resistant to cardioprotection by ischemic preconditioning. DJ-1 KO hearts showed increased mitochondrial fragmentation on electron microscopy, although there were no differences in calcium-induced MPTP opening, mitochondrial respiratory function or myocardial ATP levels. We demonstrate that loss of DJ-1 protects the heart from acute IRI cell death by preventing mitochondrial dysfunction. We propose that DJ-1 may represent a novel therapeutic target for cardioprotection.

Rationale Preconditioning is widely known to protect cardiomyocytes from reperfusion-induced cell death by activation of several pro-survival transductional pathways. The fact that preconditioning can be achieved remotely (Remote Ischaemic Preconditioning, RIPC) means that humoral factors are released from ischaemic limbs into the circulation carrying a pro-survival message. Exosomes are circulating nano-sized vesicles that mediate inter-cellular communication by carrying diverse proteins and RNA molecules. Here we studied the role of exosomes in mediating RIPC. Methods and Results We isolated exosomes from plasma of rats or humans subjected to RIPC. We characterised control or RIPC exosomes by electron microscopy, flow cytometry, western blot and nano-particle tracking analysis. Exosome concentration increased dramatically after RIPC in humans (from 3.5 ± 0.3x108 to 1.1 ± 0.3x109 exosomes/ml plasma; p < 0.01, n = 6), and administration of purified exosomes protected the heart from infarct in different settings including an in vivo rat model (vehicle: 47.4 ± 4.7; RIPC-Exosomes: 20.5 ± 3.9%Infarct/AAR; p < 0.01), ex vivo Langendorff (vehicle: 35.2 ± 3.3; RIPC-Exosomes: 21.2 ± 2.5% Infarct/AAR; p < 0.01), and in vitro hypoxia-reoxygenation of cardiomyocytes (43 ± 7% protection from death, p < 0.01). RIPC-Exosomes triggered rapid ERK phosphorylation (3.9 ± 0.1 fold over vehicle), and inhibition of upstream PI3K or MEK abolished ERK activation and inhibited cardioprotection. Conclusions We demonstrate that RIPC dramatically increases the concentration of exosomes in the circulation. Exosomes acutely activate pro-survival kinases that rapidly prepare the heart against ischemia-reperfusion injury. Exosomes represent a novel agent with the potential to be an endogenous, non-immunogenic and multi-signalling tool for cardioprotection.

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