Safety of drug use in patients with a primary mitochondrial disease: An international Delphi-based consensus.

Clinical guidance is often sought when prescribing drugs for patients with primary mitochondrial disease. Theoretical considerations concerning drug safety in patients with mitochondrial disease may lead to unnecessary withholding of a drug in a situation of clinical need. The aim of this study was to develop consensus on safe medication use in patients with a primary mitochondrial disease. A panel of 16 experts in mitochondrial medicine, pharmacology, and basic science from six different countries was established. A modified Delphi technique was used to allow the panellists to consider draft recommendations anonymously in two Delphi rounds with predetermined levels of agreement. This process was supported by a review of the available literature and a consensus conference that included the panellists and representatives of patient advocacy groups. A high level of consensus was reached regarding the safety of all 46 reviewed drugs, with the knowledge that the risk of adverse events is influenced both by individual patient risk factors and choice of drug or drug class. This paper details the consensus guidelines of an expert panel and provides an important update of previously established guidelines in safe medication use in patients with primary mitochondrial disease. Specific drugs, drug groups, and clinical or genetic conditions are described separately as they require special attention. It is important to emphasise that consensus-based information is useful to provide guidance, but that decisions related to drug prescribing should always be tailored to the specific needs and risks of each individual patient. We aim to present what is current knowledge and plan to update this regularly both to include new drugs and to review those currently included.

Cardioprotective effects of idebenone do not involve ROS scavenging: Evidence for mitochondrial complex I bypass in ischemia/reperfusion injury.

Novel therapeutic strategies to treat mitochondrial deficiencies in acute coronary syndromes are needed. Complex I of the mitochondrial electron transport system is damaged following ischemia/reperfusion (I/R) injury. This disruption contributes to aberrant electron transport, diminished bioenergetics, an altered redox environment, and mitochondrial damage involved in tissue injury. In this study, we determined the cardiac and mitochondrial effects of idebenone, a benzoquinone currently in several clinical trials with purported 'antioxidant' effects. We employed complimentary models of ischemia/reperfusion injury in perfused hearts, permeabilized cardiac fibers, isolated mitochondria, and in cells to elucidate idebenone's cardioprotective mechanism(s). In ex vivo whole hearts, infarct size was markedly reduced with post-ischemic idebenone treatment (25 ±â€¯5% area at risk, AAR) compared to controls (56 ±â€¯6% AAR, P < .05). Several parameters of hemodynamic function were also significantly improved after idebenone treatment. Parallel studies of anoxia/reoxygenation were conducted using isolated mitochondria and permeabilized ventricular fibers. In isolated mitochondria, we simultaneously monitored respiration and ROS emission. Idebenone treatment modestly elevated succinate-derived H2O2 production when compared to vehicle control (1.34 ±â€¯0.05 vs 1.21 ±â€¯0.05%, H2O2/O2 respectively, P < .05). Isolated mitochondria subjected to anoxia/reoxygenation demonstrated higher rates of respiration with idebenone treatment (2360 ±â€¯69 pmol/s*mg) versus vehicle control (1995 ±â€¯101 pmol/s*mg). Both mitochondria and permeabilized cardiac fibers produced high rates of H2O2 after anoxia/reoxygenation, with idebenone showing no discernable attenuation on H2O2 production. These insights were further investigated with studies in mitochondria isolated from reperfused ventricle. The profound decrease in complex-I dependent respiration after ischemia/reperfusion (701 ±â€¯59 pmolO2/s*mg compared to 1816 ±â€¯105 pmol O2/s*mg in normoxic mitochondria) was attenuated with idebenone treatment (994 ±â€¯76 vs pmol O2/s*mg, P < .05). Finally, the effects of idebenone were determined using permeabilized cell models with chemical inhibition of complex I. ADP-dependent oxidative phosphorylation capacity was significantly higher in complex-I inhibited cells treated acutely with idebenone (89.0 ±â€¯4.2 pmol/s*million cells versus 70.1 ±â€¯8.2 pmol/s*million cells in untreated cells). Taken together, these data indicate that the cardioprotective effects of idebenone treatment do not involve ROS-scavenging but appear to involve augmentation of the quinone pool, thus providing reducing equivalents downstream of complex I. As this compound is already in clinical trials for other indications, it may provide a safe and useful approach to mitigate ischemia/reperfusion injury in patients.

Influence of Pulsed Electric Fields and Mitochondria-Cytoskeleton Interactions on Cell Respiration.

Pulsed electric fields with microsecond pulse width (µsPEFs) are used clinically; namely, irreversible electroporation/Nanoknife is used for soft tissue tumor ablation. The µsPEF pulse parameters used in irreversible electroporation (0.5-1 kV/cm, 80-100 pulses, ∼100 µs each, 1 Hz frequency) may cause an internal field to develop within the cell because of the disruption of the outer cell membrane and subsequent penetration of the electric field. An internal field may disrupt voltage-sensitive mitochondria, although the research literature has been relatively unclear regarding whether such disruptions occur with µsPEFs. This investigation reports the influence of clinically used µsPEF parameters on mitochondrial respiration in live cells. Using a high-throughput Agilent Seahorse machine, it was observed that µsPEF exposure comprising 80 pulses with amplitudes of 600 or 700 V/cm did not alter mitochondrial respiration in 4T1 cells measured after overnight postexposure recovery. To record alterations in mitochondrial function immediately after µsPEF exposure, high-resolution respirometry was used to measure the electron transport chain state via responses to glutamate-malate and ADP and mitochondrial membrane potential via response to carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. In addition to measuring immediate mitochondrial responses to µsPEF exposure, measurements were also made on cells permeabilized using digitonin and those with compromised cytoskeleton due to actin depolymerization via treatment with the drug latrunculin B. The former treatment was used as a control to tease out the effects of plasma membrane permeabilization, whereas the latter was used to investigate indirect effects on the mitochondria that may occur if µsPEFs impact the cytoskeleton on which the mitochondria are anchored. Based on the results, it was concluded that within the pulse parameters tested, µsPEFs alone do not hinder mitochondrial physiology but can be used to impact the mitochondria upon compromising the actin. Mitochondrial susceptibility to µsPEF after actin depolymerization provides, to our knowledge, a novel avenue for cancer therapeutics.

Cardioprotective Effects of Mitochondria-Targeted Peptide SBT-20 in two Different Models of Rat Ischemia/Reperfusion.

PURPOSE: Dysfunctional mitochondria are considered to be the major source of intracellular reactive oxygen species and play a central role in the pathophysiology of myocardial ischemia/reperfusion. This study sought to determine effects of mitochondria-targeted cytoprotective peptide SBT-20 on myocardial infarct size in two different models of ischemia/reperfusion. METHODS: For in vivo studies, anesthetized Sprague Dawley rats were subjected to 30 min of coronary artery occlusion followed by 3 h of reperfusion. Rats received saline (control), low dose SBT-20 (0.3 mg/kg/h) or high dose SBT-20 (3 mg/kg/h) treatment (n = 15 rats in each group). Saline or SBT-20 were delivered into the jugular vein starting 5 min after coronary artery occlusion and were continued for one hour post coronary artery reperfusion. Body temperature, heart rate and blood pressure were monitored during the procedure. At the end of 3 h reperfusion, the ischemic risk area, no-reflow area, and infarct size were measured. In separate in vitro studies, isolated rat hearts were exposed to 20 min global ischemia, followed by SBT-20 administration (1 µM) or no SBT-20 (control) throughout the 2 h reperfusion. In vitro studies were conducted in cells and heart mitochondria to ascertain the mitochondrial effects of SBT-20 on mitochondrial respiration and reactive oxygen species production. RESULTS: In the in vivo study, the ischemic risk areas (as a percentage of the left ventricle) were similar among the saline (49.5 ± 2.3 %), low dose SBT-20 (48.6 ± 2.1 %), and high dose SBT-20 groups (48.7 ± 3.0 %). Treatment with SBT-20 significantly reduced infarct size ( as a percentage of risk area) in low dose (62.1 ± 4.4 %) and high dose (64.0 ± 4.9 %) compared with saline treatment (77.6 ± 2.6 %, p = 0.001 for both doses). There was no difference in infarct size between low and high dose SBT-20 treatment. The no-reflow areas (as a percentage of the risk area) were comparable among the saline (23.9 ± 1.7 %), low dose SBT-20 (23.7 ± 2.8 %), and high dose groups (25.0 ± 2.1 %). Body temperature, heart rate and blood pressure were comparable among the 3 groups at baseline, during ischemia, and at the end of 3 h of reperfusion. In the in vitro study, infarct size was reduced from 43.3 ± 2.6 % in control group (n = 11) to 17.2 ± 2.8 % in the SBT-20 treatment group (n = 5, p < 0.05). There were no benefits of SBT-20 on recovery of left ventricular developed pressure, coronary flow, or maximal rates of contraction/relaxation. In cell studies, treatment with SBT-20 significantly improved maximal mitochondrial respiration in response to an H2O2 challenge. In isolated mitochondria, reactive oxygen species production was significantly blunted following treatment with SBT-20. CONCLUSIONS: In summary, SBT-20 significantly reduced infarct size in two different models of myocardial injury, but did not affect hemodynamics or no-reflow area in rat heart. The reduction in injury is postulated to involve stabilization of mitochondrial function and reduced mitochondrial production of ROS.

Multimodality assessment of heart failure with preserved ejection fraction skeletal muscle reveals differences in the machinery of energy fuel metabolism.

AIMS: Skeletal muscle (SkM) abnormalities may impact exercise capacity in patients with heart failure with preserved ejection fraction (HFpEF). We sought to quantify differences in SkM oxidative phosphorylation capacity (OxPhos), fibre composition, and the SkM proteome between HFpEF, hypertensive (HTN), and healthy participants. METHODS AND RESULTS: Fifty-nine subjects (20 healthy, 19 HTN, and 20 HFpEF) performed a maximal-effort cardiopulmonary exercise test to define peak oxygen consumption (VO2, peak ), ventilatory threshold (VT), and VO2 efficiency (ratio of total work performed to O2 consumed). SkM OxPhos was assessed using Creatine Chemical-Exchange Saturation Transfer (CrCEST, n = 51), which quantifies unphosphorylated Cr, before and after plantar flexion exercise. The half-time of Cr recovery (t1/2, Cr ) was taken as a metric of in vivo SkM OxPhos. In a subset of subjects (healthy = 13, HTN = 9, and HFpEF = 12), percutaneous biopsy of the vastus lateralis was performed for myofibre typing, mitochondrial morphology, and proteomic and phosphoproteomic analysis. HFpEF subjects demonstrated lower VO2,peak , VT, and VO2 efficiency than either control group (all P < 0.05). The t1/2, Cr was significantly longer in HFpEF (P = 0.005), indicative of impaired SkM OxPhos, and correlated with cycle ergometry exercise parameters. HFpEF SkM contained fewer Type I myofibres (P = 0.003). Proteomic analyses demonstrated (a) reduced levels of proteins related to OxPhos that correlated with exercise capacity and (b) reduced ERK signalling in HFpEF. CONCLUSIONS: Heart failure with preserved ejection fraction patients demonstrate impaired functional capacity and SkM OxPhos. Reductions in the proportions of Type I myofibres, proteins required for OxPhos, and altered phosphorylation signalling in the SkM may contribute to exercise intolerance in HFpEF.

Progression-Mediated Changes in Mitochondrial Morphology Promotes Adaptation to Hypoxic Peritoneal Conditions in Serous Ovarian Cancer.

Ovarian cancer is the deadliest gynecological cancer in women, with a survival rate of less than 30% when the cancer has spread throughout the peritoneal cavity. Aggregation of cancer cells increases their viability and metastatic potential; however, there are limited studies that correlate these functional changes to specific phenotypic alterations. In this study, we investigated changes in mitochondrial morphology and dynamics during malignant transition using our MOSE cell model for progressive serous ovarian cancer. Mitochondrial morphology was changed with increasing malignancy from a filamentous network to single, enlarged organelles due to an imbalance of mitochondrial dynamic proteins (fusion: MFN1/OPA1, fission: DRP1/FIS1). These phenotypic alterations aided the adaptation to hypoxia through the promotion of autophagy and were accompanied by changes in the mitochondrial ultrastructure, mitochondrial membrane potential, and the regulation of reactive oxygen species (ROS) levels. The tumor-initiating cells increased mitochondrial fragmentation after aggregation and exposure to hypoxia that correlated well with our previously observed reduced growth and respiration in spheroids, suggesting that these alterations promote viability in non-permissive conditions. Our identification of such mitochondrial phenotypic changes in malignancy provides a model in which to identify targets for interventions aimed at suppressing metastases.

The cardiolipin-binding peptide elamipretide mitigates fragmentation of cristae networks following cardiac ischemia reperfusion in rats.

Mitochondrial dysfunction contributes to cardiac pathologies. Barriers to new therapies include an incomplete understanding of underlying molecular culprits and a lack of effective mitochondria-targeted medicines. Here, we test the hypothesis that the cardiolipin-binding peptide elamipretide, a clinical-stage compound under investigation for diseases of mitochondrial dysfunction, mitigates impairments in mitochondrial structure-function observed after rat cardiac ischemia-reperfusion. Respirometry with permeabilized ventricular fibers indicates that ischemia-reperfusion induced decrements in the activity of complexes I, II, and IV are alleviated with elamipretide. Serial block face scanning electron microscopy used to create 3D reconstructions of cristae ultrastructure reveals that disease-induced fragmentation of cristae networks are improved with elamipretide. Mass spectrometry shows elamipretide did not protect against the reduction of cardiolipin concentration after ischemia-reperfusion. Finally, elamipretide improves biophysical properties of biomimetic membranes by aggregating cardiolipin. The data suggest mitochondrial structure-function are interdependent and demonstrate elamipretide targets mitochondrial membranes to sustain cristae networks and improve bioenergetic function.

Common gut microbial metabolites of dietary flavonoids exert potent protective activities in ß-cells and skeletal muscle cells.

Flavonoids are dietary compounds with potential anti-diabetes activities. Many flavonoids have poor bioavailability and thus low circulating concentrations. Unabsorbed flavonoids are metabolized by the gut microbiota to smaller metabolites, which are more bioavailable than their precursors. The activities of these metabolites may be partly responsible for associations between flavonoids and health. However, these activities remain poorly understood. We investigated bioactivities of flavonoid microbial metabolites [hippuric acid (HA), homovanillic acid (HVA), and 5-phenylvaleric acid (5PVA)] in primary skeletal muscle and ß-cells compared to a native flavonoid [(-)-epicatechin, EC]. In muscle, EC was the most potent stimulator of glucose oxidation, while 5PVA and HA simulated glucose metabolism at 25 µM, and all compounds preserved mitochondrial function after insult. However, EC and the metabolites did not uncouple mitochonndrial respiration, with the exception of 5PVA at10 µM. In ß-cells, all metabolites more potently enhanced glucose-stimulated insulin secretion (GSIS) compared to EC. Unlike EC, the metabolites appear to enhance GSIS without enhancing ß-cell mitochondrial respiration or increasing expression of mitochondrial electron transport chain components, and with varying effects on ß-cell insulin content. The present results demonstrate the activities of flavonoid microbial metabolites for preservation of ß-cell function and glucose utilization. Additionally, our data suggest that metabolites and native compounds may act by distinct mechanisms, suggesting complementary and synergistic activities in vivo which warrant further investigation. This raises the intriguing prospect that bioavailability of native dietary flavonoids may not be as critical of a limiting factor to bioactivity as previously thought.

Expert consensus document: Mitochondrial function as a therapeutic target in heart failure.

Heart failure is a pressing worldwide public-health problem with millions of patients having worsening heart failure. Despite all the available therapies, the condition carries a very poor prognosis. Existing therapies provide symptomatic and clinical benefit, but do not fully address molecular abnormalities that occur in cardiomyocytes. This shortcoming is particularly important given that most patients with heart failure have viable dysfunctional myocardium, in which an improvement or normalization of function might be possible. Although the pathophysiology of heart failure is complex, mitochondrial dysfunction seems to be an important target for therapy to improve cardiac function directly. Mitochondrial abnormalities include impaired mitochondrial electron transport chain activity, increased formation of reactive oxygen species, shifted metabolic substrate utilization, aberrant mitochondrial dynamics, and altered ion homeostasis. In this Consensus Statement, insights into the mechanisms of mitochondrial dysfunction in heart failure are presented, along with an overview of emerging treatments with the potential to improve the function of the failing heart by targeting mitochondria.