Investigation of mitochondrial targets for cardioprotection

Background Novel therapeutic strategies are required to protect the myocardium against ischaemia- reperfusion injury. Mitochondria are fundamental for the maintenance of cell function and survival in this setting. Although a number of mitochondrial therapeutic targets have been identified, their cardioprotective efficacy in vivo remains unknown. Methods and Results This thesis describes the establishment and validation of an important in vivo murine model of ischaemia-reperfusion injury for the first time within our laboratory. This model was used to investigate the cardioprotective potential of modulating three mitochondrial proteins. In addition, this thesis investigates the use of a novel cardiac MRI sequence to quantify myocardial area-at-risk, an essential pre-requisite for assessing the cardioprotective efficacy of therapeutic interventions. Cyclophilin-D: Cyp-D has been implicated as a critical regulator of cell death by mitochondrial permeability transition pore opening upon ischaemia-reperfusion. Cyp-D genetic ablation evoked cardioprotection against myocardial ischaemia-reperfusion following extended reperfusion; thereby confirming the protective efficacy of this target. Sirtuin-3: Post-translational protein deacetylation by Sirt-3 is an important regulator of mitochondrial biology and cell survival. Sirt-3 genetic ablation did not affect the susceptibility to myocardial infarction upon ischaemia-reperfusion under normal conditions. Fasting-induced Sirt-3 overexpression increased myocardial infarct size; thereby suggesting that Sirt-3 inhibition may be protective under certain conditions. DJ-1: Preservation of mitochondrial function upon oxidative stress by the diverse actions of DJ-1 is critical for cell survival upon cerebral ischaemia-reperfusion. DJ-1 genetic ablation caused increased mitochondrial fragmentation but did not adversely affect cardiac function. DJ-1 knockout mice were more susceptible to myocardial infarction suggesting that this may represent an important target for cardioprotection. Conclusions This thesis confirms the role for CypD as an important target for cardioprotection, and provides experimental evidence implicating Sirt-3 and DJ-1 as novel mitochondrial targets for protecting the heart against ischaemia-reperfusion injury. We also provide initial data that arterial spin labelling cardiac MRI may be an important tool for the future assessment of novel cardioprotective interventions

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 Ca(2+)-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 Ca(2+) 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-1(L166P) and DJ-1(Cys106A) 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

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

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

The potential for ischemic preconditioning to reduce infarct size was first recognized more than 30 years ago. Despite extension of the concept to ischemic postconditioning and remote ischemic conditioning and literally thousands of experimental studies in various species and models which identified a multitude of signaling steps, so far there is only a single and very recent study, which has unequivocally translated cardioprotection to improved clinical outcome as the primary endpoint in patients. Many potential reasons for this disappointing lack of clinical translation of cardioprotection have been proposed, including lack of rigor and reproducibility in preclinical studies, and poor design and conduct of clinical trials. There is, however, universal agreement that robust preclinical data are a mandatory prerequisite to initiate a meaningful clinical trial. In this context, it is disconcerting that the CAESAR consortium (Consortium for preclinicAl assESsment of cARdioprotective therapies) in a highly standardized multi-center approach of preclinical studies identified only ischemic preconditioning, but not nitrite or sildenafil, when given as adjunct to reperfusion, to reduce infarct size. However, ischemic preconditioning—due to its very nature—can only be used in elective interventions, and not in acute myocardial infarction. Therefore, better strategies to identify robust and reproducible strategies of cardioprotection, which can subsequently be tested in clinical trials must be developed. We refer to the recent guidelines for experimental models of myocardial ischemia and infarction, and aim to provide now practical guidelines to ensure rigor and reproducibility in preclinical and clinical studies on cardioprotection. In line with the above guideline, we define rigor as standardized state-of-the-art design, conduct and reporting of a study, which is then a prerequisite for reproducibility, i.e. replication of results by another laboratory when performing exactly the same experiment

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

10.1038/cddis.2014.41Cell Death and Disease52e108