Rk Dongworth

Hearts deficient in both Mfn1 and Mfn2 are protected against acute myocardial infarction

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.

DJ-1 protects against cell death following acute cardiac ischemia–reperfusion injury

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.

Role of the MPTP in conditioning the heart – translatability and mechanism

Mitochondria have long been known to be the gatekeepers of cell fate. This is particularly so in the response to acute ischaemia‐reperfusion injury (IRI). Following an acute episode of sustained myocardial ischaemia, the opening of the mitochondrial permeability transition pore (MPTP) in the first few minutes of reperfusion, mediates cell death. Preventing MPTP opening at the onset of reperfusion using either pharmacological inhibitors [such as cyclosporin A (CsA) ] or genetic ablation has been reported to reduce myocardial infarct (MI) size in animal models of acute IRI. Interestingly, the endogenous cardioprotective intervention of ischaemic conditioning, in which the heart is protected against MI by applying cycles of brief ischaemia and reperfusion to either the heart itself or a remote organ or tissue, appears to be mediated through the inhibition of MPTP opening at reperfusion. Small proof‐of‐concept clinical studies have demonstrated the translatability of this therapeutic approach to target MPTP opening using CsA in clinical settings of acute myocardial IRI. However, given that CsA is a not a specific MPTP inhibitor, more novel and specific inhibitors of the MPTP need to be discovered – the molecular identification of the MPTP should facilitate this. In this paper, we review the role of the MPTP as a target for cardioprotection, the potential mechanisms underlying MPTP inhibition in the setting of ischaemic conditioning, and the translatability of MPTP inhibition as a therapeutic approach in the clinical setting.

Akt protects the heart against ischaemia-reperfusion injury by modulating mitochondrial morphology

The mechanism through which the protein kinase Akt (also called PKB), protects the heart against acute ischaemia-reperfusion injury (IRI) is not clear. Here, we investigate whether Akt mediates its cardioprotective effect by modulating mitochondrial morphology. Transfection of HL-1 cardiac cells with constitutively active Akt (caAkt) changed mitochondrial morphology as evidenced by an increase in the proportion of cells displaying predominantly elongated mitochondria (73 ± 5.0 % caAkt vs 49 ± 5.8 % control: N=80 cells/group; p< 0.05). This effect was associated with delayed time taken to induce mitochondrial permeability transition pore (MPTP) opening (by 2.4 ± 0.5 fold; N=80 cells/group: p< 0.05); and reduced cell death following simulated IRI (32.8 ± 1.2 % caAkt vs 63.8 ± 5.6 % control: N=320 cells/group: p< 0.05). Similar effects on mitochondrial morphology, MPTP opening, and cell survival post-IRI, were demonstrated with pharmacological activation of Akt using the known cardioprotective cytokine, erythropoietin (EPO). The effect of Akt on inducing mitochondrial elongation was found to be dependent on the mitochondrial fusion protein, Mitofusin-1 (Mfn1), as ablation of Mfn1 in mouse embryonic fibroblasts (MEFs) abrogated Akt-mediated mitochondrial elongation. Finally, in vivo pre-treatment with EPO reduced myocardial infarct size (as a % of the area at risk) in adult mice subjected to IRI (26.2 ± 2.6 % with EPO vs 46.1 ± 6.5 % in control; N=7/group: p< 0.05), and reduced the proportion of cells displaying myofibrillar disarray and mitochondrial fragmentation observed by electron microscopy in adult murine hearts subjected to ischaemia from 5.8 ± 1.0 % to 2.2 ± 1.0 % (N=5 hearts/group; p< 0.05). In conclusion, we found that either genetic or pharmacological activation of Akt protected the heart against acute ischaemia-reperfusion injury by modulating mitochondrial morphology.

Targeting mitochondria for cardioprotection: examining the benefit for patients

Mitochondria are critical for sustaining life, not only as the essential powerhouses of cells but as critical mediators of cell survival and death. Mitochondrial dysfunction has been identified as a key perturbation underlying numerous pathologies including myocardial ischemia-reperfusion injury and the subsequent development of impaired left ventricular systolic function and compensatory cardiac hypertrophy. This article outlines the role of mitochondrial dysfunction in these important cardiac pathologies and highlights current cardioprotective strategies and their clinical efficacy in acute myocardial infarction and heart failure patients. Finally, we explore novel mitochondrial targets and evaluate their potential future translation for clinical cardioprotection.

Quantifying the area-at-risk of myocardial infarction in-vivo using arterial spin labeling cardiac magnetic resonance

T2-weighted cardiovascular magnetic resonance (T2-CMR) of myocardial edema can quantify the area-at-risk (AAR) following acute myocardial infarction (AMI), and has been used to assess myocardial salvage by new cardioprotective therapies. However, some of these therapies may reduce edema, leading to an underestimation of the AAR by T2-CMR. Here, we investigated arterial spin labeling (ASL) perfusion CMR as a novel approach to quantify the AAR following AMI. Adult B6sv129-mice were subjected to in vivo left coronary artery ligation for 30 minutes followed by 72 hours reperfusion. T2-mapping was used to quantify the edema-based AAR (% of left ventricle) following ischemic preconditioning (IPC) or cyclosporin-A (CsA) treatment. In control animals, the AAR by T2-mapping corresponded to that delineated by histology. As expected, both IPC and CsA reduced MI size. However, IPC, but not CsA, also reduced myocardial edema leading to an underestimation of the AAR by T2-mapping. In contrast, regions of reduced myocardial perfusion delineated by cardiac ASL were able to delineate the AAR when compared to both T2-mapping and histology in control animals, and were not affected by either IPC or CsA. Therefore, ASL perfusion CMR may be an alternative method for quantifying the AAR following AMI, which unlike T2-mapping, is not affected by IPC.

CARDIAC ARTERIAL SPIN LABELLING MRI AS A NOVEL APPROACH FOR IN VIVO QUANTIFICATION OF THE AREA-AT-RISK

Rationale In order to assess the cardioprotective efficacy of a novel therapy for reducing myocardial infarct (MI) size, a reliable in vivo measure of the area-at-risk (AAR) is required. Although T2-weighted cardiac MRI (T2-MRI), which detects myocardial oedema, has been used to measure the AAR, some cardioprotective therapies have been found to reduce the extent of oedema, thereby interfering with the measured AAR by T2-MRI. Here, we investigate cardiac arterial spin labelling (ASL)-MRI as an alternative method for in vivo measuring the AAR following acute myocardial infarction. Methodology and Results B6sv129 mice (10–14 weeks old) were subjected to an in vivo left main coronary artery ligation for 30 minutes followed by 72 hours reperfusion after which MRI was performed to retrospectively measure the AAR. MRI showed regions of elevated T2 signal corresponding to the AAR delineated by Evans Blue staining (AAR/LV%: T2-MRI 60.4±2.4 versus histology 64.3±2.5: N=6/group; P>0.05). However, in ischaemic preconditioned (IPC)-treated animals, T2-MRI significantly underestimated the AAR (AAR/LV%: T2-MRI 46.0±13.2 versus histology 59.8±7.8: N=10/group; P<0.05), as IPC has reduced the extent of myocardial oedema. Interestingly, ASL-MRI revealed regions of attenuated ASL signal (indicating reduced myocardial blood flow) corresponding to the AAR delineated by Evans Blue (AAR/LV%: ASL-MRI 60.6±4.3 versus histology 64.3±3.9: N=6/group; P>0.05). Crucially, the AAR measured by ASL-MRI was not affected by IPC (AAR/LV%: ASL-MRI 53.4±7.9 versus histology 58.0±3.0: N=8/group; P>0.05). Conclusions Here we show cardiac ASL-MRI to be a novel approach for in vivo measuring the AAR following acute myocardial infarction, which unlike T2-MRI is not affected by the cardioprotective intervention, IPC.

THE ADULT MURINE HEART IS PROTECTED AGAINST ISCHEMIA-REPERFUSION INJURY IN THE ABSENCE OF BOTH MITOFUSIN (MFN) PROTEINS

Background Cardiac-specific ablation of both Mfn1 and Mfn2 in the adult heart results in mitochondrial fragmentation and a lethal cardiomyopathy after about 6 weeks. The effect of combined Mfn1 and Mfn2 deletion on the susceptibility to acute ischemia-reperfusion injury (IRI) and subsequent calcium overload is not known, and is investigated in this study. Methods and Results Cardiac-specific ablation of both Mfn1 and Mfn2 (DKO) was initiated in mice aged 5 weeks using 5 days administration of tamoxifen (MerCreMer), resulting in total knockout of both these proteins at the age of 10 weeks. These mice were subjected to in vivo myocardial ischemia (30 mins) followed by 24 hrs reperfusion before myocardial infarct size was determined. The sustained MI size in the DKO mice was 50% smaller than that in the WT control mice. These findings were associated with decreased MPTP opening susceptibility (assessed by calcium-induced mitochondrial swelling), reduced mitochondrial calcium overload after simulated IR (assessed by Rhod2 staining) and impaired mitochondrial respiration in the DKO hearts when compared to WT control. Conclusions We have shown that the adult murine heart deficient in both Mfn1 and Mfn2 was protected against acute IRI, a finding which was associated with defects in mitochondrial function and reduced mitochondrial calcium overload. This data suggests that the Mitofusins may be therapeutic targets for cardioprotection.

36 Cyclophilin-D ablation offers long-term protection against acute myocardial ischaemia-reperfusion injury

Rationale The mitochondrial permeability transition pore (mPTP) is a critical mediator of lethal myocardial reperfusion injury. Genetic ablation of cyclophilin-D (CypD, a component of the mPTP) has been reported to acutely reduce myocardial infarct size (IS) after 2 h reperfusion. However, the role of CypD ablation in long-term cardioprotection after 72 h reperfusion has not been examined. Whether IS enlarges with increasing periods of reperfusion over 72 h, to reflect the presence of ongoing lethal myocardial reperfusion injury, is controversial. Methodology Using B6sv129 mice, our first objective was to establish for the first time in our laboratory an in vivo recovery model of acute myocardial ischaemia-reperfusion injury (IRI) comprising 30 min occlusion of the left anterior descending (LAD) artery followed by extended reperfusion for 2, 6, 24 and 72 h. Infarct size was expressed as a % of the area-at-risk (IS/AAR%). Ischaemic preconditioning (IPC, comprising 5 min of LAD ischaemia/reperfusion prior to IRI) was used as a positive control. Mice deficient in CypD (CypD−/−) and wild-type littermates (CypD+/+) were subjected to IRI. Results There was no increase in IS/AAR% as the reperfusion time was prolonged over the 72 h period (38.7±4.0% at 2 h, 37.3±1.7% at 6 h, 30.5±2.5% at 24 h, 33.0±4.2% at 72 h: p>0.05: N>4/group). As expected, IPC significantly reduced IS/AAR% after 72 h reperfusion (33.0±4.2% in control vs 16.2±2.7% with IPC: p<0.05: N=6/group). Mice deficient in CypD sustained smaller IS/AAR% (35.3±4.8% in CypD+/+ vs 24.3±2.0%: p=0.05: N=8/group). Conclusions Myocardial infarct size did not enlarge with increasing duration of reperfusion. Genetic ablation of CypD confers long-term protection against IRI.