4-Hydroxynonenal impairs miRNA maturation in heart failure via Dicer post-translational modification.

BACKGROUND AND AIMS: Developing novel therapies to battle the global public health burden of heart failure remains challenging. This study investigates the underlying mechanisms and potential treatment for 4-hydroxynonenal (4-HNE) deleterious effects in heart failure. METHODS: Biochemical, functional, and histochemical measurements were applied to identify 4-HNE adducts in rat and human failing hearts. In vitro studies were performed to validate 4-HNE targets. RESULTS: 4-HNE, a reactive aldehyde by-product of mitochondrial dysfunction in heart failure, covalently inhibits Dicer, an RNase III endonuclease essential for microRNA (miRNA) biogenesis. 4-HNE inhibition of Dicer impairs miRNA processing. Mechanistically, 4-HNE binds to recombinant human Dicer through an intermolecular interaction that disrupts both activity and stability of Dicer in a concentration- and time-dependent manner. Dithiothreitol neutralization of 4-HNE or replacing 4-HNE-targeted residues in Dicer prevents 4-HNE inhibition of Dicer in vitro. Interestingly, end-stage human failing hearts from three different heart failure aetiologies display defective 4-HNE clearance, decreased Dicer activity, and miRNA biogenesis impairment. Notably, boosting 4-HNE clearance through pharmacological re-activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) using Alda-1 or its improved orally bioavailable derivative AD-9308 restores Dicer activity. ALDH2 is a major enzyme responsible for 4-HNE removal. Importantly, this response is accompanied by improved miRNA maturation and cardiac function/remodelling in a pre-clinical model of heart failure. CONCLUSIONS: 4-HNE inhibition of Dicer directly impairs miRNA biogenesis in heart failure. Strikingly, decreasing cardiac 4-HNE levels through pharmacological ALDH2 activation is sufficient to re-establish Dicer activity and miRNA biogenesis; thereby representing potential treatment for patients with heart failure.

Mitochondrial Fusion, Fission, and Mitophagy in Cardiac Diseases: Challenges and Therapeutic Opportunities.

Significance: Mitochondria play a critical role in the physiology of the heart by controlling cardiac metabolism, function, and remodeling. Accumulation of fragmented and damaged mitochondria is a hallmark of cardiac diseases. Recent Advances: Disruption of quality control systems that maintain mitochondrial number, size, and shape through fission/fusion balance and mitophagy results in dysfunctional mitochondria, defective mitochondrial segregation, impaired cardiac bioenergetics, and excessive oxidative stress. Critical Issues: Pharmacological tools that improve the cardiac pool of healthy mitochondria through inhibition of excessive mitochondrial fission, boosting mitochondrial fusion, or increasing the clearance of damaged mitochondria have emerged as promising approaches to improve the prognosis of heart diseases. Future Directions: There is a reasonable amount of preclinical evidence supporting the effectiveness of molecules targeting mitochondrial fission and fusion to treat cardiac diseases. The current and future challenges are turning these lead molecules into treatments. Clinical studies focusing on acute (i.e., myocardial infarction) and chronic (i.e., heart failure) cardiac diseases are needed to validate the effectiveness of such strategies in improving mitochondrial morphology, metabolism, and cardiac function. Antioxid. Redox Signal. 36, 844-863.

Activation of PKCε-ALDH2 Axis Prevents 4-HNE-Induced Pain in Mice.

Protein kinase Cε (PKCε) is highly expressed in nociceptor neurons and its activation has been reported as pro-nociceptive. Intriguingly, we previously demonstrated that activation of the mitochondrial PKCε substrate aldehyde dehydrogenase-2 (ALDH2) results in anti-nociceptive effects. ALDH2 is a major enzyme responsible for the clearance of 4-hydroxy-2-nonenal (4-HNE), an oxidative stress byproduct accumulated in inflammatory conditions and sufficient to induce pain hypersensitivity in rodents. Here we determined the contribution of the PKCε-ALDH2 axis during 4-HNE-induced mechanical hypersensitivity. Using knockout mice, we demonstrated that PKCε is essential for the nociception recovery during 4-HNE-induced hypersensitivity. We also found that ALDH2 deficient knockin mice display increased 4-HNE-induced nociceptive behavior. As proof of concept, the use of a selective peptide activator of PKCε (ΨεHSP90), which favors PKCε translocation to mitochondria and activation of PKCε-ALDH2 axis, was sufficient to block 4-HNE-induced hypersensitivity in WT, but not in ALDH2-deficient mice. Similarly, ΨεHSP90 administration prevented mechanical hypersensitivity induced by endogenous production of 4-HNE after carrageenan injection. These findings provide evidence that selective activation of mitochondrial PKCε-ALDH2 axis is important to mitigate aldehyde-mediated pain in rodents, suggesting that ΨεHSP90 and small molecules that mimic it may be a potential treatment for patients with pain.

Activation of PKCε-ALDH2 axis prevents 4-HNE-induced pain in mice / A ativação do eixo PKCε-ALDH2 previne a dor induzida por 4-HNE em camundongos

Protein kinase Cε (PKCε) is highly expressed in nociceptor neurons and its activation has been reported as pro-nociceptive. Intriguingly, we previously demonstrated that activation of the mitochondrial PKCε substrate aldehyde dehydrogenase-2 (ALDH2) results in anti-nociceptive effects. ALDH2 is a major enzyme responsible for the clearance of 4-hydroxy-2-nonenal (4-HNE), an oxidative stress byproduct accumulated in inflammatory conditions and sufficient to induce pain hypersensitivity in rodents. Here we determined the contribution of the PKCε-ALDH2 axis during 4-HNE-induced mechanical hypersensitivity. Using knockout mice, we demonstrated that PKCε is essential for the nociception recovery during 4-HNE-induced hypersensitivity. We also found that ALDH2 deficient knockin mice display increased 4-HNE-induced nociceptive behavior. As proof of concept, the use of a selective peptide activator of PKCε (ΨεHSP90), which favors PKCε translocation to mitochondria and activation of PKCε-ALDH2 axis, was sufficient to block 4-HNE-induced hypersensitivity in WT, but not in ALDH2-deficient mice. Similarly, ΨεHSP90 administration prevented mechanical hypersensitivity induced by endogenous production of 4-HNE after carrageenan injection. These findings provide evidence that selective activation of mitochondrial PKCε-ALDH2 axis is important to mitigate aldehyde-mediated pain in rodents, suggesting that ΨεHSP90 and small molecules that mimic it may be a potential treatment for patients with pain.

Activation of PKCε-ALDH2 axis prevents 4-HNE-induced pain in mice

Protein kinase Cε (PKCε) is highly expressed in nociceptor neurons and its activation has been reported as pro-nociceptive. Intriguingly, we previously demonstrated that activation of the mitochondrial PKCε substrate aldehyde dehydrogenase-2 (ALDH2) results in anti-nociceptive effects. ALDH2 is a major enzyme responsible for the clearance of 4-hydroxy-2-nonenal (4-HNE), an oxidative stress byproduct accumulated in inflammatory conditions and sufficient to induce pain hypersensitivity in rodents. Here we determined the contribution of the PKCε-ALDH2 axis during 4-HNE-induced mechanical hypersensitivity. Using knockout mice, we demonstrated that PKCε is essential for the nociception recovery during 4-HNE-induced hypersensitivity. We also found that ALDH2 deficient knockin mice display increased 4-HNE-induced nociceptive behavior. As proof of concept, the use of a selective peptide activator of PKCε (ΨεHSP90), which favors PKCε translocation to mitochondria and activation of PKCε-ALDH2 axis, was sufficient to block 4-HNE-induced hypersensitivity in WT, but not in ALDH2-deficient mice. Similarly, ΨεHSP90 administration prevented mechanical hypersensitivity induced by endogenous production of 4-HNE after carrageenan injection. These findings provide evidence that selective activation of mitochondrial PKCε-ALDH2 axis is important to mitigate aldehyde-mediated pain in rodents, suggesting that ΨεHSP90 and small molecules that mimic it may be a potential treatment for patients with pain.

A selective inhibitor of mitofusin 1-ßIIPKC association improves heart failure outcome in rats.

We previously demonstrated that beta II protein kinase C (ßIIPKC) activity is elevated in failing hearts and contributes to this pathology. Here we report that ßIIPKC accumulates on the mitochondrial outer membrane and phosphorylates mitofusin 1 (Mfn1) at serine 86. Mfn1 phosphorylation results in partial loss of its GTPase activity and in a buildup of fragmented and dysfunctional mitochondria in heart failure. ßIIPKC siRNA or a ßIIPKC inhibitor mitigates mitochondrial fragmentation and cell death. We confirm that Mfn1-ßIIPKC interaction alone is critical in inhibiting mitochondrial function and cardiac myocyte viability using SAMßA, a rationally-designed peptide that selectively antagonizes Mfn1-ßIIPKC association. SAMßA treatment protects cultured neonatal and adult cardiac myocytes, but not Mfn1 knockout cells, from stress-induced death. Importantly, SAMßA treatment re-establishes mitochondrial morphology and function and improves cardiac contractility in rats with heart failure, suggesting that SAMßA may be a potential treatment for patients with heart failure.

Targeting mitochondrial dysfunction and oxidative stress in heart failure: Challenges and opportunities.

Mitochondrial dysfunction characterized by impaired bioenergetics, oxidative stress and aldehydic load is a hallmark of heart failure. Recently, different research groups have provided evidence that selective activation of mitochondrial detoxifying systems that counteract excessive accumulation of ROS, RNS and reactive aldehydes is sufficient to stop cardiac degeneration upon chronic stress, such as heart failure. Therefore, pharmacological and non-pharmacological approaches targeting mitochondria detoxification may play a critical role in the prevention or treatment of heart failure. In this review we discuss the most recent findings on the central role of mitochondrial dysfunction, oxidative stress and aldehydic load in heart failure, highlighting the most recent preclinical and clinical studies using mitochondria-targeted molecules and exercise training as effective tools against heart failure.

Cardioprotection induced by a brief exposure to acetaldehyde: role of aldehyde dehydrogenase 2.

Aims: We previously demonstrated that acute ethanol administration protects the heart from ischaemia/reperfusion (I/R) injury thorough activation of aldehyde dehydrogenase 2 (ALDH2). Here, we characterized the role of acetaldehyde, an intermediate product from ethanol metabolism, and its metabolizing enzyme, ALDH2, in an ex vivo model of cardiac I/R injury. Methods and results: We used a combination of homozygous knock-in mice (ALDH2*2), carrying the human inactivating point mutation ALDH2 (E487K), and a direct activator of ALDH2, Alda-1, to investigate the cardiac effect of acetaldehyde. The ALDH2*2 mice have impaired acetaldehyde clearance, recapitulating the human phenotype. Yet, we found a similar infarct size in wild type (WT) and ALDH2*2 mice. Similar to ethanol-induced preconditioning, pre-treatment with 50 µM acetaldehyde increased ALDH2 activity and reduced cardiac injury in hearts of WT mice without affecting cardiac acetaldehyde levels. However, acetaldehyde pre-treatment of hearts of ALDH2*2 mice resulted in a three-fold increase in cardiac acetaldehyde levels and exacerbated I/R injury. Therefore, exogenous acetaldehyde appears to have a bimodal effect in I/R, depending on the ALDH2 genotype. Further supporting an ALDH2 role in cardiac preconditioning, pharmacological ALDH2 inhibition abolished ethanol-induced cardioprotection in hearts of WT mice, whereas a selective activator, Alda-1, protected ALDH2*2 against ethanol-induced cardiotoxicity. Finally, either genetic or pharmacological inhibition of ALDH2 mitigated ischaemic preconditioning. Conclusion: Taken together, our findings suggest that low levels of acetaldehyde are cardioprotective whereas high levels are damaging in an ex vivo model of I/R injury and that ALDH2 is a major, but not the only, regulator of cardiac acetaldehyde levels and protection from I/R.

Exercise reestablishes autophagic flux and mitochondrial quality control in heart failure.

We previously reported that facilitating the clearance of damaged mitochondria through macroautophagy/autophagy protects against acute myocardial infarction. Here we characterize the impact of exercise, a safe strategy against cardiovascular disease, on cardiac autophagy and its contribution to mitochondrial quality control, bioenergetics and oxidative damage in a post-myocardial infarction-induced heart failure animal model. We found that failing hearts displayed reduced autophagic flux depicted by accumulation of autophagy-related markers and loss of responsiveness to chloroquine treatment at 4 and 12 wk after myocardial infarction. These changes were accompanied by accumulation of fragmented mitochondria with reduced O2 consumption, elevated H2O2 release and increased Ca2+-induced mitochondrial permeability transition pore opening. Of interest, disruption of autophagic flux was sufficient to decrease cardiac mitochondrial function in sham-treated animals and increase cardiomyocyte toxicity upon mitochondrial stress. Importantly, 8 wk of exercise training, starting 4 wk after myocardial infarction at a time when autophagy and mitochondrial oxidative capacity were already impaired, improved cardiac autophagic flux. These changes were followed by reduced mitochondrial number:size ratio, increased mitochondrial bioenergetics and better cardiac function. Moreover, exercise training increased cardiac mitochondrial number, size and oxidative capacity without affecting autophagic flux in sham-treated animals. Further supporting an autophagy mechanism for exercise-induced improvements of mitochondrial bioenergetics in heart failure, acute in vivo inhibition of autophagic flux was sufficient to mitigate the increased mitochondrial oxidative capacity triggered by exercise in failing hearts. Collectively, our findings uncover the potential contribution of exercise in restoring cardiac autophagy flux in heart failure, which is associated with better mitochondrial quality control, bioenergetics and cardiac function.

Disruption of mitochondrial quality control in peripheral artery disease: New therapeutic opportunities.

Peripheral artery disease (PAD) is a multifactorial disease initially triggered by reduced blood supply to the lower extremities due to atherosclerotic obstructions. It is considered a major public health problem worldwide, affecting over 200 million people. Management of PAD includes smoking cessation, exercise, statin therapy, antiplatelet therapy, antihypertensive therapy and surgical intervention. Although these pharmacological and non-pharmacological interventions usually increases blood flow to the ischemic limb, morbidity and mortality associated with PAD continue to increase. This scenario raises new fundamental questions regarding the contribution of intrinsic metabolic changes in the distal affected skeletal muscle to the progression of PAD. Recent evidence suggests that disruption of skeletal muscle mitochondrial quality control triggered by intermittent ischemia-reperfusion injury is associated with increased morbidity in individuals with PAD. The mitochondrial quality control machinery relies on surveillance systems that help maintaining mitochondrial homeostasis upon stress. In this review, we describe some of the most critical mechanisms responsible for the impaired skeletal muscle mitochondrial quality control in PAD. We also discuss recent findings on the central role of mitochondrial bioenergetics and quality control mechanisms including mitochondrial fusion-fission balance, turnover, oxidative stress and aldehyde metabolism in the pathophysiology of PAD, and highlight their potential as therapeutic targets.

Aldehydic load and aldehyde dehydrogenase 2 profile during the progression of post-myocardial infarction cardiomyopathy: benefits of Alda-1.

BACKGROUND/OBJECTIVES: We previously demonstrated that reducing cardiac aldehydic load by aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme responsible for metabolizing the major lipid peroxidation product, protects against acute ischemia/reperfusion injury and chronic heart failure. However, time-dependent changes in ALDH2 profile, aldehydic load and mitochondrial bioenergetics during progression of post-myocardial infarction (post-MI) cardiomyopathy are unknown and should be established to determine the optimal time window for drug treatment. METHODS: Here we characterized cardiac ALDH2 activity and expression, lipid peroxidation, 4-hydroxy-2-nonenal (4-HNE) adduct formation, glutathione pool and mitochondrial energy metabolism and H2O2 release during the 4 weeks after permanent left anterior descending (LAD) coronary artery occlusion in rats. RESULTS: We observed a sustained disruption of cardiac mitochondrial function during the progression of post-MI cardiomyopathy, characterized by >50% reduced mitochondrial respiratory control ratios and up to 2 fold increase in H2O2 release. Mitochondrial dysfunction was accompanied by accumulation of cardiac and circulating lipid peroxides and 4-HNE protein adducts and down-regulation of electron transport chain complexes I and V. Moreover, increased aldehydic load was associated with a 90% reduction in cardiac ALDH2 activity and increased glutathione pool. Further supporting an ALDH2 mechanism, sustained Alda-1 treatment (starting 24h after permanent LAD occlusion surgery) prevented aldehydic overload, mitochondrial dysfunction and improved ventricular function in post-MI cardiomyopathy rats. CONCLUSION: Taken together, our findings demonstrate a disrupted mitochondrial metabolism along with an insufficient cardiac ALDH2-mediated aldehyde clearance during the progression of ventricular dysfunction, suggesting a potential therapeutic value of ALDH2 activators during the progression of post-myocardial infarction cardiomyopathy.

Aldehyde dehydrogenase-2 regulates nociception in rodent models of acute inflammatory pain.

Exogenous aldehydes can cause pain in animal models, suggesting that aldehyde dehydrogenase-2 (ALDH2), which metabolizes many aldehydes, may regulate nociception. To test this hypothesis, we generated a knock-in mouse with an inactivating point mutation in ALDH2 (ALDH2*2), which is also present in human ALDH2 of ~540 million East Asians. The ALDH2*1/*2 heterozygotic mice exhibited a larger response to painful stimuli than their wild-type littermates, and this heightened nociception was inhibited by an ALDH2-selective activator (Alda-1). No effect on inflammation per se was observed. Using a rat model, we then showed that nociception tightly correlated with ALDH activity (R(2) = 0.90) and that reduced nociception was associated with less early growth response protein 1 (EGR1) in the spinal cord and less reactive aldehyde accumulation at the insult site (including acetaldehyde and 4-hydroxynonenal). Further, acetaldehyde- and formalin-induced nociceptive behavior was greater in the ALDH2*1/*2 mice than in the wild-type mice. Finally, Alda-1 treatment was even beneficial when given after the inflammatory agent was administered. Our data in rodent models suggest that the mitochondrial enzyme ALDH2 regulates nociception and could serve as a molecular target for pain control, with ALDH2 activators, such as Alda-1, as potential non-narcotic, cardiac-safe analgesics. Furthermore, our results suggest a possible genetic basis for East Asians' apparent lower pain tolerance.

Aldehyde dehydrogenase 2 activation in heart failure restores mitochondrial function and improves ventricular function and remodelling.

AIMS: We previously demonstrated that pharmacological activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) protects the heart against acute ischaemia/reperfusion injury. Here, we determined the benefits of chronic activation of ALDH2 on the progression of heart failure (HF) using a post-myocardial infarction model. METHODS AND RESULTS: We showed that a 6-week treatment of myocardial infarction-induced HF rats with a selective ALDH2 activator (Alda-1), starting 4 weeks after myocardial infarction at a time when ventricular remodelling and cardiac dysfunction were present, improved cardiomyocyte shortening, cardiac function, left ventricular compliance and diastolic function under basal conditions, and after isoproterenol stimulation. Importantly, sustained Alda-1 treatment showed no toxicity and promoted a cardiac anti-remodelling effect by suppressing myocardial hypertrophy and fibrosis. Moreover, accumulation of 4-hydroxynonenal (4-HNE)-protein adducts and protein carbonyls seen in HF was not observed in Alda-1-treated rats, suggesting that increasing the activity of ALDH2 contributes to the reduction of aldehydic load in failing hearts. ALDH2 activation was associated with improved mitochondrial function, including elevated mitochondrial respiratory control ratios and reduced H2O2 release. Importantly, selective ALDH2 activation decreased mitochondrial Ca(2+)-induced permeability transition and cytochrome c release in failing hearts. Further supporting a mitochondrial mechanism for ALDH2, Alda-1 treatment preserved mitochondrial function upon in vitro aldehydic load. CONCLUSIONS: Selective activation of mitochondrial ALDH2 is sufficient to improve the HF outcome by reducing the toxic effects of aldehydic overload on mitochondrial bioenergetics and reactive oxygen species generation, suggesting that ALDH2 activators, such as Alda-1, have a potential therapeutic value for treating HF patients.

Peripheral sensitization increases opioid receptor expression and activation by crotalphine in rats.

Inflammation enhances the peripheral analgesic efficacy of opioid drugs, but the mechanisms involved in this phenomenon have not been fully elucidated. Crotalphine (CRP), a peptide that was first isolated from South American rattlesnake C.d. terrificus venom, induces a potent and long-lasting anti-nociceptive effect that is mediated by the activation of peripheral opioid receptors. Because the high efficacy of CRP is only observed in the presence of inflammation, we aimed to elucidate the mechanisms involved in the CRP anti-nociceptive effect induced by inflammation. Using real-time RT-PCR, western blot analysis and ELISA assays, we demonstrate that the intraplantar injection of prostaglandin E2 (PGE2) increases the mRNA and protein levels of the µ- and κ-opioid receptors in the dorsal root ganglia (DRG) and paw tissue of rats within 3 h of the injection. Using conformation state-sensitive antibodies that recognize activated opioid receptors, we show that PGE2, alone does not increase the activation of these opioid receptors but that in the presence of PGE2, the activation of specific opioid receptors by CRP and selective µ- and κ-opioid receptor agonists (positive controls) increases. Furthermore, PGE2 down-regulated the expression and activation of the δ-opioid receptor. CRP increased the level of activated mitogen-activated protein kinases in cultured DRG neurons, and this increase was dependent on the activation of protein kinase Cζ. This CRP effect was much more prominent when the cells were pretreated with PGE2. These results indicate that the expression and activation of peripheral opioid receptors by opioid-like drugs can be up- or down-regulated in the presence of an acute injury and that acute tissue injury enhances the efficacy of peripheral opioids.

Exercise training restores cardiac protein quality control in heart failure.

Exercise training is a well-known coadjuvant in heart failure treatment; however, the molecular mechanisms underlying its beneficial effects remain elusive. Despite the primary cause, heart failure is often preceded by two distinct phenomena: mitochondria dysfunction and cytosolic protein quality control disruption. The objective of the study was to determine the contribution of exercise training in regulating cardiac mitochondria metabolism and cytosolic protein quality control in a post-myocardial infarction-induced heart failure (MI-HF) animal model. Our data demonstrated that isolated cardiac mitochondria from MI-HF rats displayed decreased oxygen consumption, reduced maximum calcium uptake and elevated H2O2 release. These changes were accompanied by exacerbated cardiac oxidative stress and proteasomal insufficiency. Declined proteasomal activity contributes to cardiac protein quality control disruption in our MI-HF model. Using cultured neonatal cardiomyocytes, we showed that either antimycin A or H2O2 resulted in inactivation of proteasomal peptidase activity, accumulation of oxidized proteins and cell death, recapitulating our in vivo model. Of interest, eight weeks of exercise training improved cardiac function, peak oxygen uptake and exercise tolerance in MI-HF rats. Moreover, exercise training restored mitochondrial oxygen consumption, increased Ca²âº-induced permeability transition and reduced H2O2 release in MI-HF rats. These changes were followed by reduced oxidative stress and better cardiac protein quality control. Taken together, our findings uncover the potential contribution of mitochondrial dysfunction and cytosolic protein quality control disruption to heart failure and highlight the positive effects of exercise training in re-establishing cardiac mitochondrial physiology and protein quality control, reinforcing the importance of this intervention as a non-pharmacological tool for heart failure therapy.