Neuromuscular Dysfunction Precedes Cognitive Impairment in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) develops along a continuum that spans years prior to diagnosis. Decreased muscle function and mitochondrial respiration occur years earlier in those that develop AD; however, it is unknown what causes these peripheral phenotypes in a disease of the brain. Exercise promotes muscle, mitochondria, and cognitive health and is proposed to be a potential therapeutic for AD, but no study has investigated how skeletal muscle adapts to exercise training in an AD-like context. Utilizing 5xFAD mice, an AD model that develops ad-like pathology and cognitive impairments around 6 mo of age, we examined in vivo neuromuscular function and exercise adapations (mitochondrial respiration and RNA sequencing) before the manifestation of overt cognitive impairment. We found 5xFAD mice develop neuromuscular dysfunction beginning as early as 4 mo of age, characterized by impaired nerve-stimulated muscle torque production and compound nerve action potential of the sciatic nerve. Furthermore, skeletal muscle in 5xFAD mice had altered, sex-dependent, adaptive responses (mitochondrial respiration and gene expression) to exercise training in the absence of overt cognitive impairment. Changes in peripheral systems, specifically neural communication to skeletal muscle, may be harbingers for AD and have implications for lifestyle interventions, like exercise, in AD.

Evaluation and Treatment of Acute Trauma Pain in Older Adults.

In the context of an ageing population, the demographic sands of trauma are shifting. Increasingly, trauma units are serving older adults who have sustained injuries in low-energy falls from a standing height. Older age is commonly associated with changes in physiology, as well as an increased prevalence of frailty and multimorbidity, including cardiac, renal and liver disease. These factors can complicate the safe and effective administration of analgesia in the older trauma patient. Trauma services therefore need to adapt to meet this demographic shift and ensure that trauma clinicians are sufficiently skilled in treating pain in complex older people. This article is dedicated to the management of acute trauma pain in older adults. It aims to highlight the notable clinical challenges of managing older trauma patients compared with their younger counterparts. It offers an overview of the evidence and practical opinion on the merits and drawbacks of commonly used analgesics, as well as more novel and emerging analgesic adjuncts. A search of Medline (Ovid, from inception to 7 November 2022) was conducted by a medical librarian to identify relevant articles using keyword and subject heading terms for trauma, pain, older adults and analgesics. Results were limited to articles published in the last 10 years and English language. Relevant articles' references were hand-screened to identify other relevant articles. There is paucity of dedicated high-quality evidence to guide management of trauma-related pain in older adults. Ageing-related changes in physiology, the accumulation of multimorbidity, frailty and the risk of inducing delirium secondary to analgesic medication present a suite of challenges in the older trauma patient. An important nuance of treating pain in older trauma patients is the challenge of balancing iatrogenic adverse effects of analgesia against the harms of undertreated pain, the complications and consequences of which include immobility, pneumonia, sarcopenia, pressure ulcers, long-term functional decline, increased long-term care needs and mortality. In this article, the role of non-opioid agents including short-course non-steroidal anti-inflammatory drugs (NSAIDs) is discussed. Opioid selection and dosing are reviewed for older adults suffering from acute trauma pain in the context of kidney and liver disease. The evidence base and limitations of other adjuncts such as topical and intravenous lidocaine, ketamine and regional anaesthesia in acute geriatric trauma are discussed.

Amputation and advance care plans: An observational study exploring decision making and long-term outcomes in a vascular centre.

BACKGROUND: Half of those undergoing major lower limb amputation for peripheral arterial disease die within 1 year. Advance care planning reduces days in hospital and increases the chance of dying in a preferred place. AIM: To investigate the prevalence and content of advance care planning for people having a lower limb amputation due to acute or chronic limb-threatening ischaemia or diabetes. Secondary aims were to explore its association with mortality, and length of hospital stay. DESIGN: A retrospective observational cohort study. The intervention was advance care planning. SETTING/PARTICIPANTS: Patients admitted to the South West England Major Arterial Centre between 1 January 2019 and 1 January 2021 who received unilateral or bilateral below, above, or through knee amputation due to acute or chronic limb-threatening ischaemia or diabetes. RESULTS: 116 patients were included in the study. 20.7% (n = 24) died within 1 year. 40.5% (n = 47) had an advance care planning discussion of which all included cardiopulmonary resuscitation decisions with few exploring other options. Patients who were more likely to have advance care planning discussions were ≥75 years (aOR = 5.58, 95%CI 1.56-20.0), female (aOR = 3.24, 95%CI 1.21-8.69), and had multimorbidity (Charlson Comorbidity Index ≥5, aOR = 2.97, 95%CI 1.11-7.92). Discussions occurred more often in the emergency pathway and were predominantly initiated by physicians. Advance care planning was associated with increased mortality (aHR = 2.63, 95%CI 1.01, 5.02) and longer hospital stay (aHR = 0.52, 95%CI 0.32-0.83). CONCLUSIONS: Despite a high risk of death for all patients in the months following amputation, advance care planning occurred in fewer than half of people and mostly focused on resuscitation.

Protocol for the development of a multidisciplinary clinical practice guideline for the care of patients with chronic subdural haematoma.

Introduction: A common neurosurgical condition, chronic subdural haematoma (cSDH) typically affects older people with other underlying health conditions. The care of this potentially vulnerable cohort is often, however, fragmented and suboptimal. In other complex conditions, multidisciplinary guidelines have transformed patient experience and outcomes, but no such framework exists for cSDH. This paper outlines a protocol to develop the first comprehensive multidisciplinary guideline from diagnosis to long-term recovery with cSDH. Methods: The project will be guided by a steering group of key stakeholders and professional organisations and will feature patient and public involvement. Multidisciplinary thematic working groups will examine key aspects of care to formulate appropriate, patient-centered research questions, targeted with evidence review using the GRADE framework. The working groups will then formulate draft clinical recommendations to be used in a modified Delphi process to build consensus on guideline contents. Conclusions: We present a protocol for the development of a multidisciplinary guideline to inform the care of patients with a cSDH, developed by cross-disciplinary working groups and arrived at through a consensus-building process, including a modified online Delphi.

Electronic cigarette vaping with aged coils causes acute lung injury in mice.

Electronic cigarettes (e-cigarettes) have been used widely as an alternative to conventional cigarettes and have become particularly popular among young adults. A growing body of evidence has shown that e-cigarettes are associated with acute lung injury and adverse effects in multiple other organs. Previous studies showed that high emissions of aldehydes (formaldehyde and acetaldehyde) in aerosols were associated with increased usage of the same e-cigarette coils. However, the impact on lung function of using aged coils has not been reported. We investigated the relationship between coil age and acute lung injury in mice exposed to experimental vaping for 1 h (2 puffs/min, 100 ml/puff). The e-liquid contains propylene glycol and vegetable glycerin (50:50, vol) only. The concentrations of formaldehyde and acetaldehyde in the vaping aerosols increased with age of the nichrome coils starting at 1200 puffs. Mice exposed to e-cigarette aerosols produced from 1800, but not 0 or 900, puff-aged coils caused acute lung injury, increased lung wet/dry weight ratio, and induced lung inflammation (IL-6, TNF-α, IL-1ß, MIP-2). Exposure to vaping aerosols from 1800 puff-aged coils decreased heart rate, respiratory rate, and oxygen saturation in mice compared to mice exposed to air or aerosols from new coils. In conclusion, we observed that the concentration of aldehydes (formaldehyde and acetaldehyde) increased with repeated and prolonged usage of e-cigarette coils. Exposure to high levels of aldehyde in vaping aerosol was associated with acute lung injury in mice. These findings show significant risk of lung injury associated with prolonged use of e-cigarette devices.

Dietary Conjugated Linoleic Acid Reduces Body Weight and Fat in Snord116m+/p- and Snord116m-/p- Mouse Models of Prader-Willi Syndrome.

Prader-Willi Syndrome (PWS) is a human genetic condition that affects up to 1 in 10,000 live births. Affected infants present with hypotonia and developmental delay. Hyperphagia and increasing body weight follow unless drastic calorie restriction is initiated. Recently, our laboratory showed that one of the genes in the deleted locus causative for PWS, Snord116, maintains increased expression of hypothalamic Nhlh2, a basic helix-loop-helix transcription factor. We have previously also shown that obese mice with a deletion of Nhlh2 respond to a conjugated linoleic acid (CLA) diet with weight and fat loss. In this study, we investigated whether mice with a paternal deletion of Snord116 (Snord116m+/p-) would respond similarly. We found that while Snord116m+/p- mice and mice with a deletion of both Snord116 alleles were not significantly obese on a high-fat diet, they did lose body weight and fat on a high-fat/CLA diet, suggesting that the genotype did not interfere with CLA actions. There were no changes in food intake or metabolic rate, and only moderate differences in exercise performance. RNA-seq and microbiome analyses identified hypothalamic mRNAs, and differentially populated gut bacteria, that support future mechanistic analyses. CLA may be useful as a food additive to reduce obesity in humans with PWS.

Multidrug resistance proteins preferentially regulate natriuretic peptide-driven cGMP signalling in the heart and vasculature.

BACKGROUND AND PURPOSE: cGMP underpins the bioactivity of NO and natriuretic peptides and is key to cardiovascular homeostasis. cGMP-driven responses are terminated primarily by PDEs, but cellular efflux via multidrug resistance proteins (MRPs) might contribute. Herein, the effect of pharmacological blockade of MRPs on cGMP signalling in the heart and vasculature was investigated in vitro and in vivo. EXPERIMENTAL APPROACH: Proliferation of human coronary artery smooth muscle cells (hCASMCs), vasorelaxation of murine aorta and reductions in mean arterial BP (MABP) in response to NO donors or natriuretic peptides were determined in the absence and presence of the MRP inhibitor MK571. The ability of MRP inhibition to reverse morphological and contractile deficits in a murine model of pressure overload-induced heart failure was also explored. KEY RESULTS: MK571 attenuated hCASMC growth and enhanced the anti-proliferative effects of NO and atrial natriuretic peptide (ANP). MRP blockade caused concentration-dependent relaxations of murine aorta and augmented responses to ANP (and to a lesser extent NO). MK571 did not decrease MABP per se but enhanced the hypotensive actions of ANP and improved structural and functional indices of disease severity in experimental heart failure. These beneficial actions of MRP inhibition were associated with a greater intracellular:extracellular cGMP ratio in vitro and in vivo. CONCLUSIONS AND IMPLICATIONS: MRP blockade promotes the cardiovascular functions of natriuretic peptides in vitro and in vivo, with more modest effects on NO. MRP inhibition may have therapeutic utility in cardiovascular diseases triggered by dysfunctional cGMP signalling, particularly those associated with altered natriuretic peptide bioactivity. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Erratum: Voluntary wheel running complements microdystrophin gene therapy to improve muscle function in mdx mice.

[This corrects the article DOI: 10.1016/j.omtm.2021.02.024.].

Sulfide catabolism ameliorates hypoxic brain injury.

The mammalian brain is highly vulnerable to oxygen deprivation, yet the mechanism underlying the brain's sensitivity to hypoxia is incompletely understood. Hypoxia induces accumulation of hydrogen sulfide, a gas that inhibits mitochondrial respiration. Here, we show that, in mice, rats, and naturally hypoxia-tolerant ground squirrels, the sensitivity of the brain to hypoxia is inversely related to the levels of sulfide:quinone oxidoreductase (SQOR) and the capacity to catabolize sulfide. Silencing SQOR increased the sensitivity of the brain to hypoxia, whereas neuron-specific SQOR expression prevented hypoxia-induced sulfide accumulation, bioenergetic failure, and ischemic brain injury. Excluding SQOR from mitochondria increased sensitivity to hypoxia not only in the brain but also in heart and liver. Pharmacological scavenging of sulfide maintained mitochondrial respiration in hypoxic neurons and made mice resistant to hypoxia. These results illuminate the critical role of sulfide catabolism in energy homeostasis during hypoxia and identify a therapeutic target for ischemic brain injury.

Voluntary wheel running complements microdystrophin gene therapy to improve muscle function in mdx mice.

We tested the hypothesis that voluntary wheel running would complement microdystrophin gene therapy to improve muscle function in young mdx mice, a model of Duchenne muscular dystrophy. mdx mice injected with a single dose of AAV9-CK8-microdystrophin or vehicle at age 7 weeks were assigned to three groups: mdxRGT (run, gene therapy), mdxGT (no run, gene therapy), or mdx (no run, no gene therapy). Wild-type (WT) mice were assigned to WTR (run) and WT (no run) groups. WTR and mdxRGT performed voluntary wheel running for 21 weeks; remaining groups were cage active. Robust expression of microdystrophin occurred in heart and limb muscles of treated mice. mdxRGT versus mdxGT mice showed increased microdystrophin in quadriceps but decreased levels in diaphragm. mdx final treadmill fatigue time was depressed compared to all groups, improved in mdxGT, and highest in mdxRGT. Both weekly running distance (km) and final treadmill fatigue time for mdxRGT and WTR were similar. Remarkably, mdxRGT diaphragm power was only rescued to 60% of WT, suggesting a negative impact of running. However, potential changes in fiber type distribution in mdxRGT diaphragms could indicate an adaptation to trade power for endurance. Post-treatment in vivo maximal plantar flexor torque relative to baseline values was greater for mdxGT and mdxRGT versus all other groups. Mitochondrial respiration rates from red quadriceps fibers were significantly improved in mdxGT animals, but the greatest bioenergetic benefit was observed in the mdxRGT group. Additional assessments revealed partial to full functional restoration in mdxGT and mdxRGT muscles relative to WT. These data demonstrate that voluntary wheel running combined with microdystrophin gene therapy in young mdx mice improved whole-body performance, affected muscle function differentially, mitigated energetic deficits, but also revealed some detrimental effects of exercise. With microdystrophin gene therapy currently in clinical trials, these data may help us understand the potential impact of exercise in treated patients.

Hypoxia ameliorates brain hyperoxia and NAD+ deficiency in a murine model of Leigh syndrome.

Leigh syndrome is a severe mitochondrial neurodegenerative disease with no effective treatment. In the Ndufs4-/- mouse model of Leigh syndrome, continuously breathing 11% O2 (hypoxia) prevents neurodegeneration and leads to a dramatic extension (~5-fold) in lifespan. We investigated the effect of hypoxia on the brain metabolism of Ndufs4-/- mice by studying blood gas tensions and metabolite levels in simultaneously sampled arterial and cerebral internal jugular venous (IJV) blood. Relatively healthy Ndufs4-/- and wildtype (WT) mice breathing air until postnatal age ~38 d were compared to Ndufs4-/- and WT mice breathing air until ~38 days old followed by 4-weeks of breathing 11% O2. Compared to WT control mice, Ndufs4-/- mice breathing air have reduced brain O2 consumption as evidenced by an elevated partial pressure of O2 in IJV blood (PijvO2) despite a normal PO2 in arterial blood, and higher lactate/pyruvate (L/P) ratios in IJV plasma revealed by metabolic profiling. In Ndufs4-/- mice, hypoxia treatment normalized the cerebral venous PijvO2 and L/P ratios, and decreased levels of nicotinate in IJV plasma. Brain concentrations of nicotinamide adenine dinucleotide (NAD+) were lower in Ndufs4-/- mice breathing air than in WT mice, but preserved at WT levels with hypoxia treatment. Although mild hypoxia (17% O2) has been shown to be an ineffective therapy for Ndufs4-/- mice, we find that when combined with nicotinic acid supplementation it provides a modest improvement in neurodegeneration and lifespan. Therapies targeting both brain hyperoxia and NAD+ deficiency may hold promise for treating Leigh syndrome.

A novel murine muscle loading model to investigate Achilles musculotendinous adaptation.

Achilles tendinopathy is a debilitating condition affecting the entire spectrum of society and a condition that increases the risk of tendon rupture. Effective therapies remain elusive, as anti-inflammatory drugs and surgical interventions show poor long-term outcomes. Eccentric loading of the Achilles muscle-tendon unit is an effective physical therapy for treatment of symptomatic human tendinopathy. Here, we introduce a novel mouse model of hindlimb muscle loading designed to achieve a tissue-targeted therapeutic exercise. This model includes the application of tissue (muscle and tendon)-loading "doses," coupled with ankle dorsiflexion and plantarflexion, inspired by human clinical protocols. Under computer control, the foot was rotated through the entire ankle joint range of motion while the plantar flexors simultaneously contracted to simulate body mass loading, consistent with human therapeutic exercises. This approach achieved two key components of the heel drop and raise movement: ankle range of motion coupled with body mass loading. Model development entailed the tuning of parameters such as footplate speed, number of repetitions, number of sets of repetitions, treatment frequency, treatment duration, and treatment timing. Initial model development was carried out on uninjured mice to define a protocol that was well tolerated and nondeleterious to tendon biomechanical function. When applied to a murine Achilles tendinopathy model, muscle loading led to a significant improvement in biomechanical outcome measures, with a decrease in cross-sectional area and an increase in material properties, compared with untreated animals. Our model facilitates the future investigation of mechanisms whereby rehabilitative muscle loading promotes healing of Achilles tendon injuries.NEW & NOTEWORTHY We introduce a novel mouse model of hindlimb muscle loading designed to achieve a tissue-targeted therapeutic exercise. This innovative model allows for application of muscle loading "doses," coupled with ankle dorsiflexion and plantarflexion, inspired by human loading clinical treatment. Our model facilitates future investigation of mechanisms whereby rehabilitative muscle loading promotes healing of Achilles tendon injuries.

Airway stem cells sense hypoxia and differentiate into protective solitary neuroendocrine cells.

Neuroendocrine (NE) cells are epithelial cells that possess many of the characteristics of neurons, including the presence of secretory vesicles and the ability to sense environmental stimuli. The normal physiologic functions of solitary airway NE cells remain a mystery. We show that mouse and human airway basal stem cells sense hypoxia. Hypoxia triggers the direct differentiation of these stem cells into solitary NE cells. Ablation of these solitary NE cells during hypoxia results in increased epithelial injury, whereas the administration of the NE cell peptide CGRP rescues this excess damage. Thus, we identify stem cells that directly sense hypoxia and respond by differentiating into solitary NE cells that secrete a protective peptide that mitigates hypoxic injury.

Potential cross-talk between muscle and tendon in Duchenne muscular dystrophy.

PURPOSE: To describe potential signaling (cross-talk) between dystrophic skeletal muscle and tendon in Duchenne muscular dystrophy. MATERIALS AND METHODS: Review of Duchenne muscular dystrophy and associated literature relevant to muscle-tendon cross-talk. RESULTS AND CONCLUSIONS: Duchenne muscular dystrophy results from the absence of the protein dystrophin and the associated dystrophin - glycoprotein complex, which are thought to provide both structural support and signaling functions for the muscle fiber. In addition, there are other potential signal pathways that could represent cross-talk between muscle and tendon, particularly at the myotendinous junction. Duchenne muscular dystrophy is characterized by multiple pathophysiologic mechanisms. Herein, we explore three of these: (1) the extracellular matrix, fibrosis, and fat deposition; (2) satellite cells; and (3) tensegrity. A key signaling protein that emerged in each was transforming growth factor - beta one (TGF-ß1).].

A novel ex vivo protocol to mimic human walking gait: implications for Duchenne muscular dystrophy.

We developed a novel ex vivo mouse protocol to mimic in vivo human soleus muscle function predicted by musculoskeletal simulations to better understand eccentric contractions during gait and ultimately to better understand their effects in Duchenne muscular dystrophy (DMD) muscles. DMD muscles are susceptible to eccentric injury because the protein dystrophin is absent. The mdx mouse, a DMD model that also lacks dystrophin, is often subjected to ex vivo acute but nonphysiological eccentric injury protocols. It is possible these acute protocols either over- or underestimate eccentric stresses and strains compared with those from humans during gait. To explore this possibility, healthy human soleus excitation, force, and length change profiles during a single walking stride (gait cycle) were simulated using OpenSim and then scaled to an ex vivo mouse soleus preparation based on muscle architectural measurements. Aurora Scientific, Inc., software and a 701C electrical stimulator were modified to discretely modulate muscle stimulation voltage at constant frequency and finely control muscle length changes to produce a force pattern that correctly mimicked the gait cycle from simulations. In a proof-of-principle study, wild-type and mdx mice soleus muscles were subjected to 25 gait cycles. Modest fatigue was evident in the muscles at the 25th versus first gait cycle for both genotypes, but both rapidly recovered isometric force within 1 min of the last cycle. These data indicate that the ex vivo gait protocol was well tolerated. More important, this protocol provides a novel assessment tool to determine the effects of physiological eccentric contractions on dystrophic muscle.NEW & NOTEWORTHY A novel ex vivo mouse soleus protocol that mimics scaled length change and excitation profiles predicted by a mathematical model of human soleus during gait is presented. A custom stimulator was developed that enabled an innovative muscle stimulation technique to modulate voltage to closely match the excitation pattern of human soleus during gait. This ex vivo protocol provides assessment of simulated human movement in mouse muscle, including components of eccentric contractions.

Laminin-111 protein therapy after disease onset slows muscle disease in a mouse model of laminin-α2 related congenital muscular dystrophy.

Laminin-α2 related congenital muscular dystrophy (LAMA2-CMD) is a fatal muscle disease caused by mutations in the LAMA2 gene. Laminin-α2 is critical for the formation of laminin-211 and -221 heterotrimers in the muscle basal lamina. LAMA2-CMD patients exhibit hypotonia from birth and progressive muscle loss that results in developmental delay, confinement to a wheelchair, respiratory insufficiency and premature death. There is currently no cure or effective treatment for LAMA2-CMD. Several studies have shown laminin-111 can serve as an effective protein-replacement therapy for LAMA2-CMD. Studies have demonstrated early treatment with laminin-111 protein results in an increase in life expectancy and improvements in muscle pathology and function. Since LAMA2-CMD patients are often diagnosed after advanced disease, it is unclear if laminin-111 protein therapy at an advanced stage of the disease can have beneficial outcomes. In this study, we tested the efficacy of laminin-111 protein therapy after disease onset in a mouse model of LAMA2-CMD. Our results showed laminin-111 treatment after muscle disease onset increased life expectancy, promoted muscle growth and increased muscle stiffness. Together these studies indicate laminin-111 protein therapy either early or late in the disease process could serve as an effective protein replacement therapy for LAMA2-CMD.

Pro-opiomelanocortin Neurons and the Transcriptional Regulation of Motivated Exercise.

Hypothalamic pro-opiomelanocortin (POMC) neurons are key sensory neurons for energy balance. The basic helix-loop-helix transcription factor NHLH2 is expressed in POMC neurons, and Nhlh2 knockout mice show adult-onset obesity with low exercise behavior. Evidence is presented to explore the hypothesis that NHLH2 transcriptional activity within POMC neurons is crucial for maintaining motivated spontaneous activity and enforced exercise.

AAV-Mediated Gene Transfer Restores a Normal Muscle Transcriptome in a Canine Model of X-Linked Myotubular Myopathy.

Multiple clinical trials employing recombinant adeno-associated viral (rAAV) vectors have been initiated for neuromuscular disorders, including Duchenne and limb-girdle muscular dystrophies, spinal muscular atrophy, and recently X-linked myotubular myopathy (XLMTM). Our previous work on a canine model of XLMTM showed that a single rAAV8-cMTM1 systemic infusion corrected structural abnormalities within the muscle and restored contractile function, with affected dogs surviving more than 4 years post injection. This remarkable therapeutic efficacy presents a unique opportunity to identify the downstream molecular drivers of XLMTM pathology and to what extent the whole muscle transcriptome is restored to normal after gene transfer. Herein, RNA-sequencing was used to examine the transcriptomes of the Biceps femoris and Vastus lateralis in a previously described canine cohort that showed dose-dependent clinical improvements after rAAV8-cMTM1 gene transfer. Our analysis confirmed several dysregulated genes previously observed in XLMTM mice but also identified transcripts linked to XLMTM pathology. We demonstrated XLMTM transcriptome remodeling and dose-dependent normalization of gene expression after gene transfer and created metrics to pinpoint potential biomarkers of disease progression and correction.

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.