A novel small molecule inhibitor of human Drp1.

Mitochondrial dynamin-related protein 1 (Drp1) is a large GTPase regulator of mitochondrial dynamics and is known to play an important role in numerous pathophysiological processes. Despite being the most widely used Drp1 inhibitor, the specificity of Mdivi-1 towards human Drp1 has not been definitively proven and there have been numerous issues reported with its use including off-target effects. In our hands Mdivi-1 showed varying binding affinities toward human Drp1, potentially impacted by compound aggregation. Herein, we sought to identify a novel small molecule inhibitor of Drp1. From an initial virtual screening, we identified DRP1i27 as a compound which directly bound to the human isoform 3 of Drp1 via surface plasmon resonance and microscale thermophoresis. Importantly, DRP1i27 was found to have a dose-dependent increase in the cellular networks of fused mitochondria but had no effect in Drp1 knock-out cells. Further analogues of this compound were identified and screened, though none displayed greater affinity to human Drp1 isoform 3 than DRP1i27. To date, this is the first small molecule inhibitor shown to directly bind to human Drp1.

Hydralazine protects the heart against acute ischaemia/reperfusion injury by inhibiting Drp1-mediated mitochondrial fission.

AIMS: Genetic and pharmacological inhibition of mitochondrial fission induced by acute myocardial ischaemia/reperfusion injury (IRI) has been shown to reduce myocardial infarct size. The clinically used anti-hypertensive and heart failure medication, hydralazine, is known to have anti-oxidant and anti-apoptotic effects. Here, we investigated whether hydralazine confers acute cardioprotection by inhibiting Drp1-mediated mitochondrial fission. METHODS AND RESULTS: Pre-treatment with hydralazine was shown to inhibit both mitochondrial fission and mitochondrial membrane depolarisation induced by oxidative stress in HeLa cells. In mouse embryonic fibroblasts (MEFs), pre-treatment with hydralazine attenuated mitochondrial fission and cell death induced by oxidative stress, but this effect was absent in MEFs deficient in the mitochondrial fission protein, Drp1. Molecular docking and surface plasmon resonance studies demonstrated binding of hydralazine to the GTPase domain of the mitochondrial fission protein, Drp1 (KD 8.6±1.0 µM), and inhibition of Drp1 GTPase activity in a dose-dependent manner. In isolated adult murine cardiomyocytes subjected to simulated IRI, hydralazine inhibited mitochondrial fission, preserved mitochondrial fusion events, and reduced cardiomyocyte death (hydralazine 24.7±2.5% vs. control 34.1±1.5%, P=0.0012). In ex vivo perfused murine hearts subjected to acute IRI, pre-treatment with hydralazine reduced myocardial infarct size (as % left ventricle: hydralazine 29.6±6.5% vs. vehicle control 54.1±4.9%, P=0.0083), and in the murine heart subjected to in vivo IRI, the administration of hydralazine at reperfusion, decreased myocardial infarct size (as % area-at-risk: hydralazine 28.9±3.0% vs. vehicle control 58.2±3.8%, P<0.001). CONCLUSION: We show that, in addition to its antioxidant and anti-apoptotic effects, hydralazine, confers acute cardioprotection by inhibiting IRI-induced mitochondrial fission, raising the possibility of repurposing hydralazine as a novel cardioprotective therapy for improving post-infarction outcomes.

New perspectives on the role of Drp1 isoforms in regulating mitochondrial pathophysiology.

Mitochondria are dynamic organelles constantly undergoing fusion and fission. A concerted balance between the process of mitochondrial fusion and fission is required to maintain cellular health under different physiological conditions. Mutation and dysregulation of Drp1, the major driver of mitochondrial fission, has been associated with various neurological, oncological and cardiovascular disorders. Moreover, when subjected to pathological insults, mitochondria often undergo excessive fission, generating fragmented and dysfunctional mitochondria leading to cell death. Therefore, manipulating mitochondrial fission by targeting Drp1 has been an appealing therapeutic approach for cytoprotection. However, studies have been inconsistent. Studies employing Drp1 constructs representing alternate Drp1 isoforms, have demonstrated differing impacts of these isoforms on mitochondrial fission and cell death. Furthermore, there are distinct expression patterns of Drp1 isoforms in different tissues, suggesting idiosyncratic engagement in specific cellular functions. In this review, we will discuss these inherent variations among human Drp1 isoforms and how they could affect Drp1-mediated mitochondrial fission and cell death.

Mitochondrial Assays Using Cardiac Stem Cells.

Ischemic heart disease is the leading cause of death worldwide. Stem cell therapy to repair and regenerate the infarcted myocardium is a promising approach to address this unmet medical need. However, the poor survival of transplanted cells in the hostile ischemic myocardium has been a major hurdle in achieving an effective cell therapy against myocardial infarction. As such, novel strategies to promote the survival of transplanted cells are highly sought after. Mitochondria are intimately involved in cell survival and have been the main organelles being targeted for cytoprotection. Mitochondrial morphology is linked to mitochondrial function and cell viability. Therefore, quantitative methodologies to obtain reliable and reproducible results of mitochondrial morphology and function are essential for identifying and developing new cytoprotective strategies to enhance the survival of stem cells post-transplantation. Here, we describe methods for assessing mitochondrial morphology, mitochondrial membrane potential, and mitochondrial reactive oxygen species production.

Mdivi-1 Protects Human W8B2+ Cardiac Stem Cells from Oxidative Stress and Simulated Ischemia-Reperfusion Injury.

Cardiac stem cell (CSC) therapy is a promising approach to treat ischemic heart disease. However, the poor survival of transplanted stem cells in the ischemic myocardium has been a major impediment in achieving an effective cell-based therapy against myocardial infarction. Inhibiting mitochondrial fission has been shown to promote survival of several cell types. However, the role of mitochondrial morphology in survival of human CSC remains unknown. In this study, we investigated whether mitochondrial division inhibitor-1 (Mdivi-1), an inhibitor of mitochondrial fission protein dynamin-related protein-1 (Drp1), can improve survival of a novel population of human W8B2+ CSCs in hydrogen peroxide (H2O2)-induced oxidative stress and simulated ischemia-reperfusion injury models. Mdivi-1 significantly reduced H2O2-induced cell death in a dose-dependent manner. This cytoprotective effect was accompanied by an increased proportion of cells with tubular mitochondria, but independent of mitochondrial membrane potential recovery and reduction of mitochondrial superoxide production. In simulated ischemia-reperfusion injury model, Mdivi-1 given as a pretreatment or throughout ischemia-reperfusion injury significantly reduced cell death. However, the cytoprotective effect of Mdivi-1 was not observed when given at reperfusion. Moreover, the cytoprotective effect of Mdivi-1 in the simulated ischemia-reperfusion injury model was not accompanied by changes in mitochondrial morphology, mitochondrial membrane potential, or mitochondrial reactive oxygen species production. Mdivi-1 also did not affect mitochondrial bioenergetics of intact W8B2+ CSCs. Taken together, these experiments demonstrated that Mdivi-1 treatment of human W8B2+ CSCs enhances their survival and can be employed to improve therapeutic efficacy of CSCs for ischemic heart disease.

Mitochondrial fission - a drug target for cytoprotection or cytodestruction?

Mitochondria are morphologically dynamic organelles constantly undergoing processes of fission and fusion that maintain integrity and bioenergetics of the organelle: these processes are vital for cell survival. Disruption in the balance of mitochondrial fusion and fission is thought to play a role in several pathological conditions including ischemic heart disease. Proteins involved in regulating the processes of mitochondrial fusion and fission are therefore potential targets for pharmacological therapies. Mdivi-1 is a small molecule inhibitor of the mitochondrial fission protein Drp1. Inhibiting mitochondrial fission with Mdivi-1 has proven cytoprotective benefits in several cell types involved in a wide array of cardiovascular injury models. On the other hand, Mdivi-1 can also exert antiproliferative and cytotoxic effects, particularly in hyperproliferative cells. In this review, we discuss these divergent effects of Mdivi-1 on cell survival, as well as the potential and limitations of Mdivi-1 as a therapeutic agent.

Harnessing the secretome of cardiac stem cells as therapy for ischemic heart disease.

Adult stem cells continue to promise opportunities to repair damaged cardiac tissue. However, precisely how adult stem cells accomplish cardiac repair, especially after ischemic damage, remains controversial. It has been postulated that the clinical benefit of adult stem cells for cardiovascular disease results from the release of cytokines and growth factors by the transplanted cells. Studies in animal models of myocardial infarction have reported that such paracrine factors released from transplanted adult stem cells contribute to improved cardiac function by several processes. These include promoting neovascularization of damaged tissue, reducing inflammation, reducing fibrosis and scar formation, as well as protecting cardiomyocytes from apoptosis. In addition, these factors might also stimulate endogenous repair by activating cardiac stem cells. Interestingly, stem cells discovered to be resident in the heart appear to be functionally superior to extra-cardiac adult stem cells when transplanted for cardiac repair and regeneration. In this review, we discuss the therapeutic potential of cardiac stem cells and how the proteins secreted from these cells might be harnessed to promote repair and regeneration of damaged cardiac tissue. We also highlight how recent controversies about the efficacy of adult stem cells in clinical trials of ischemic heart disease have not dampened enthusiasm for the application of cardiac stem cells and their paracrine factors for cardiac repair: the latter have proved superior to the mesenchymal stem cells used in most clinical trials in the past, some of which appear to have been conducted with sub-optimal rigor.