Aldehyde dehydrogenase 2 activation and coevolution of its εPKC-mediated phosphorylation sites.

BACKGROUND: Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a key enzyme for the metabolism of many toxic aldehydes such as acetaldehyde, derived from alcohol drinking, and 4HNE, an oxidative stress-derived lipid peroxidation aldehyde. Post-translational enhancement of ALDH2 activity can be achieved by serine/threonine phosphorylation by epsilon protein kinase C (εPKC). Elevated ALDH2 is beneficial in reducing injury following myocardial infarction, stroke and other oxidative stress and aldehyde toxicity-related diseases. We have previously identified three εPKC phosphorylation sites, threonine 185 (T185), serine 279 (S279) and threonine 412 (T412), on ALDH2. Here we further characterized the role and contribution of each phosphorylation site to the enhancement of enzymatic activity by εPKC. METHODS: Each individual phosphorylation site was mutated to a negatively charged amino acid, glutamate, to mimic a phosphorylation, or to a non-phosphorylatable amino acid, alanine. ALDH2 enzyme activities and protection against 4HNE inactivation were measured in the presence or absence of εPKC phosphorylation in vitro. Coevolution of ALDH2 and its εPKC phosphorylation sites was delineated by multiple sequence alignments among a diverse range of species and within the ALDH multigene family. RESULTS: We identified S279 as a critical εPKC phosphorylation site in the activation of ALDH2. The critical catalytic site, cysteine 302 (C302) of ALDH2 is susceptible to adduct formation by reactive aldehyde, 4HNE, which readily renders the enzyme inactive. We show that phosphomimetic mutations of T185E, S279E and T412E confer protection of ALDH2 against 4HNE-induced inactivation, indicating that phosphorylation on these three sites by εPKC likely also protects the enzyme against reactive aldehydes. Finally, we demonstrate that the three ALDH2 phosphorylation sites co-evolved with εPKC over a wide range of species. Alignment of 18 human ALDH isozymes, indicates that T185 and S279 are unique ALDH2, εPKC specific phosphorylation sites, while T412 is found in other ALDH isozymes. We further identified three highly conserved serine/threonine residues (T384, T433 and S471) in all 18 ALDH isozymes that may play an important phosphorylation-mediated regulatory role in this important family of detoxifying enzymes. CONCLUSION: εPKC phosphorylation and its coevolution with ALDH2 play an important role in the regulation and protection of ALDH2 enzyme activity.

Mitochondrial reactive oxygen species at the heart of the matter: new therapeutic approaches for cardiovascular diseases.

Reactive oxygen species (ROS) have been implicated in a variety of age-related diseases, including multiple cardiovascular disorders. However, translation of ROS scavengers (antioxidants) into the clinic has not been successful. These antioxidants grossly reduce total levels of cellular ROS including ROS that participate in physiological signaling. In this review, we challenge the traditional antioxidant therapeutic approach that targets ROS directly with novel approaches that improve mitochondrial functions to more effectively treat cardiovascular diseases.

Selective Phosphorylation Inhibitor of Delta Protein Kinase C-Pyruvate Dehydrogenase Kinase Protein-Protein Interactions: Application for Myocardial Injury in Vivo.

Protein kinases regulate numerous cellular processes, including cell growth, metabolism, and cell death. Because the primary sequence and the three-dimensional structure of many kinases are highly similar, the development of selective inhibitors for only one kinase is challenging. Furthermore, many protein kinases are pleiotropic, mediating diverse and sometimes even opposing functions by phosphorylating multiple protein substrates. Here, we set out to develop an inhibitor of a selective protein kinase phosphorylation of only one of its substrates. Focusing on the pleiotropic delta protein kinase C (δPKC), we used a rational approach to identify a distal docking site on δPKC for its substrate, pyruvate dehydrogenase kinase (PDK). We reasoned that an inhibitor of PDK's docking should selectively inhibit the phosphorylation of only PDK without affecting phosphorylation of the other δPKC substrates. Our approach identified a selective inhibitor of PDK docking to δPKC with an in vitro Kd of ∼50 nM and reducing cardiac injury IC50 of ∼5 nM. This inhibitor, which did not affect the phosphorylation of other δPKC substrates even at 1 µM, demonstrated that PDK phosphorylation alone is critical for δPKC-mediated injury by heart attack. The approach we describe is likely applicable for the identification of other substrate-specific kinase inhibitors.

Pharmacological inhibition of ßIIPKC is cardioprotective in late-stage hypertrophy.

We previously found that in the hearts of hypertensive Dahl salt-sensitive rats, ßIIPKC levels increase during the transition from compensated cardiac hypertrophy to cardiac dysfunction. Here we showed that a six-week treatment of these hypertensive rats with a ßIIPKC-specific inhibitor, ßIIV5-3, prolonged their survival by at least 6weeks, suppressed myocardial fibrosis and inflammation, and delayed the transition from compensated hypertrophy to cardiac dysfunction. In addition, changes in the levels of the Ca(2+)-handling proteins, SERCA2 and the Na(+)/Ca(2+) exchanger, as well as troponin I phosphorylation, seen in the control-treated hypertensive rats were not observed in the ßΙΙPKC-treated rats, suggesting that ßΙΙPKC contributes to the regulation of calcium levels in the myocardium. In contrast, treatment with the selective inhibitor of ßIPKC, an alternative spliced form of ßIIPKC, had no beneficial effects in these rats. We also found that ßIIV5-3, but not ßIV5-3, improved calcium handling in isolated rat cardiomyocytes and enhanced contractility in isolated rat hearts. In conclusion, our data using an in vivo model of cardiac dysfunction (late-phase hypertrophy), suggest that ßIIPKC contributes to the pathology associated with heart failure and thus an inhibitor of ßIIPKC may be a potential treatment for this disease.

Activation of aldehyde dehydrogenase 2 (ALDH2) confers cardioprotection in protein kinase C epsilon (PKCvarepsilon) knockout mice.

Acute administration of ethanol can reduce cardiac ischemia/reperfusion injury. Previous studies demonstrated that the acute cytoprotective effect of ethanol on the myocardium is mediated by protein kinase C epsilon (PKCvarepsilon). We recently identified aldehyde dehydrogenase 2 (ALDH2) as a PKCvarepsilon substrate, whose activation is necessary and sufficient to confer cardioprotection in vivo. ALDH2 metabolizes cytotoxic reactive aldehydes, such as 4-hydroxy-2-nonenal (4-HNE), which accumulate during cardiac ischemia/reperfusion. Here, we used a combination of PKCvarepsilon knockout mice and a direct activator of ALDH2, Alda-44, to further investigate the interplay between PKCvarepsilon and ALDH2 in cardioprotection. We report that ethanol preconditioning requires PKCvarepsilon, whereas direct activation of ALDH2 reduces infarct size in both wild type and PKCvarepsilon knockout hearts. Our data suggest that ALDH2 is downstream of PKCvarepsilon in ethanol preconditioning and that direct activation of ALDH2 can circumvent the requirement of PKCvarepsilon to induce cytoprotection. We also report that in addition to ALDH2 activation, Alda-44 prevents 4-HNE induced inactivation of ALDH2 by reducing the formation of 4-HNE-ALDH2 protein adducts. Thus, Alda-44 promotes metabolism of cytotoxic reactive aldehydes that accumulate in ischemic myocardium. Taken together, our findings suggest that direct activation of ALDH2 may represent a method of harnessing the cardioprotective effect of ethanol without the side effects associated with alcohol consumption.

Time-dependent and ethanol-induced cardiac protection from ischemia mediated by mitochondrial translocation of varepsilonPKC and activation of aldehyde dehydrogenase 2.

The cardioprotective effects of moderate alcohol consumption have been well documented in animal models and in humans. Protection afforded against ischemia and reperfusion injury (I/R) proceeds through an ischemic preconditioning-like mechanism involving the activation of epsilon protein kinase C (varepsilonPKC) and is dependent on the time and duration of ethanol treatment. However, the substrates of varepsilonPKC and the molecular mechanisms by which the enzyme protects the heart from oxidative damage induced by I/R are not fully described. Using an open-chest model of acute myocardial infarction in vivo, we find that intraperitoneal injection of ethanol (0.5 g/kg) 60 min prior to (but not 15 min prior to) a 30-minute transient ligation of the left anterior descending coronary artery reduced I/R-mediated injury by 57% (measured as a decrease of creatine phosphokinase release into the blood). Only under cardioprotective conditions, ethanol treatment resulted in the translocation of varepsilonPKC to cardiac mitochondria, where the enzyme bound aldehyde dehydrogenase-2 (ALDH2). ALDH2 is an intra-mitochondrial enzyme involved in the detoxification of toxic aldehydes such as 4-hydroxy-2-nonenal (4-HNE) and 4-HNE mediates oxidative damage, at least in part, by covalently modifying and inactivating proteins (by forming 4-HNE adducts). In hearts subjected to I/R after ethanol treatment, the levels of 4-HNE protein adducts were lower and JNK1/2 and ERK1/2 activities were diminished relative to the hearts from rats subjected to I/R in the absence of ethanol. Together, this work provides an insight into the mitochondrial-dependent basis of ethanol-induced and varepsilonPKC-mediated protection from cardiac ischemia, in vivo.

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.

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.

Potential biomarkers to follow the progression and treatment response of Huntington's disease.

Huntington's disease (HD) is a rare genetic disease caused by expanded polyglutamine repeats in the huntingtin protein resulting in selective neuronal loss. Although genetic testing readily identifies those who will be affected, current pharmacological treatments do not prevent or slow down disease progression. A major challenge is the slow clinical progression and the inability to biopsy the affected tissue, the brain, making it difficult to design short and effective proof of concept clinical trials to assess treatment benefit. In this study, we focus on identifying peripheral biomarkers that correlate with the progression of the disease and treatment benefit. We recently developed an inhibitor of pathological mitochondrial fragmentation, P110, to inhibit neurotoxicity in HD. Changes in levels of mitochondrial DNA (mtDNA) and inflammation markers in plasma, a product of DNA oxidation in urine, mutant huntingtin aggregates, and 4-hydroxynonenal adducts in muscle and skin tissues were all noted in HD R6/2 mice relative to wild-type mice. Importantly, P110 treatment effectively reduced the levels of these biomarkers. Finally, abnormal levels of mtDNA were also found in plasma of HD patients relative to control subjects. Therefore, we identified several potential peripheral biomarkers as candidates to assess HD progression and the benefit of intervention for future clinical trials.

Impaired GAPDH-induced mitophagy contributes to the pathology of Huntington's disease.

Mitochondrial dysfunction is implicated in multiple neurodegenerative diseases. In order to maintain a healthy population of functional mitochondria in cells, defective mitochondria must be properly eliminated by lysosomal machinery in a process referred to as mitophagy. Here, we uncover a new molecular mechanism underlying mitophagy driven by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) under the pathological condition of Huntington's disease (HD) caused by expansion of polyglutamine repeats. Expression of expanded polyglutamine tracts catalytically inactivates GAPDH (iGAPDH), which triggers its selective association with damaged mitochondria in several cell culture models of HD. Through this mechanism, iGAPDH serves as a signaling molecule to induce direct engulfment of damaged mitochondria into lysosomes (micro-mitophagy). However, abnormal interaction of mitochondrial GAPDH with long polyglutamine tracts stalled GAPDH-mediated mitophagy, leading to accumulation of damaged mitochondria, and increased cell death. We further demonstrated that overexpression of inactive GAPDH rescues this blunted process and enhances mitochondrial function and cell survival, indicating a role for GAPDH-driven mitophagy in the pathology of HD.

New therapeutics to modulate mitochondrial dynamics and mitophagy in cardiac diseases.

The processes that control the number and shape of the mitochondria (mitochondrial dynamics) and the removal of damaged mitochondria (mitophagy) have been the subject of intense research. Recent work indicates that these processes may contribute to the pathology associated with cardiac diseases. This review describes some of the key proteins that regulate these processes and their potential as therapeutic targets for cardiac diseases.

The challenge in translating basic research discoveries to treatment of Huntington disease.

Huntington disease is a rare neurodegenerative disease resulting from insertion and/or expansion of a polyglutamine repeats close to the N-terminal of the huntingtin protein. Although unequivocal genetic tests have been available for about 20 years, current pharmacological treatments do not prevent or slow down disease progression. Recent basic research identified potential novel drug targets for the treatment of Huntington disease. However, there are clear challenges in translating these discoveries into treatment strategies for these patients. The following is a brief discussion of these challenges using our recent experience as an example.

Protein folding creates structure-based, noncontiguous consensus phosphorylation motifs recognized by kinases.

Linear consensus motifs are short contiguous sequences of residues within a protein that can form recognition modules for protein interaction or catalytic modification. Protein kinase specificity and the matching of kinases to substrates have been mostly defined by phosphorylation sites that occur in linear consensus motifs. However, phosphorylation can also occur within sequences that do not match known linear consensus motifs recognized by kinases and within flexible loops. We report the identification of Thr(253) in α-tubulin as a site that is phosphorylated by protein kinase C ßI (PKCßI). Thr(253) is not part of a linear PKC consensus motif. Instead, Thr(253) occurs within a region on the surface of α-tubulin that resembles a PKC phosphorylation site consensus motif formed by basic residues in different parts of the protein, which come together in the folded protein to form the recognition motif for PKCßI. Mutations of these basic residues decreased substrate phosphorylation, confirming the presence of this "structurally formed" consensus motif and its importance for the protein kinase-substrate interaction. Analysis of previously reported protein kinase A (PKA) and PKC substrates identified sites within structurally formed consensus motifs in many substrates of these two kinase families. Thus, the concept of consensus phosphorylation site motif needs to be expanded to include sites within these structurally formed consensus motifs.

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.

Inhibition of mitochondrial fragmentation diminishes Huntington's disease-associated neurodegeneration.

Huntington's disease (HD) is the result of expression of a mutated Huntingtin protein (mtHtt), and is associated with a variety of cellular dysfunctions including excessive mitochondrial fission. Here, we tested whether inhibition of excessive mitochondrial fission prevents mtHtt-induced pathology. We developed a selective inhibitor (P110-TAT) of the mitochondrial fission protein dynamin-related protein 1 (DRP1). We found that P110-TAT inhibited mtHtt-induced excessive mitochondrial fragmentation, improved mitochondrial function, and increased cell viability in HD cell culture models. P110-TAT treatment of fibroblasts from patients with HD and patients with HD with iPS cell-derived neurons reduced mitochondrial fragmentation and corrected mitochondrial dysfunction. P110-TAT treatment also reduced the extent of neurite shortening and cell death in iPS cell-derived neurons in patients with HD. Moreover, treatment of HD transgenic mice with P110-TAT reduced mitochondrial dysfunction, motor deficits, neuropathology, and mortality. We found that p53, a stress gene involved in HD pathogenesis, binds to DRP1 and mediates DRP1-induced mitochondrial and neuronal damage. Furthermore, P110-TAT treatment suppressed mtHtt-induced association of p53 with mitochondria in multiple HD models. These data indicate that inhibition of DRP1-dependent excessive mitochondrial fission with a P110-TAT-like inhibitor may prevent or slow the progression of HD.

Acute inhibition of excessive mitochondrial fission after myocardial infarction prevents long-term cardiac dysfunction.

BACKGROUND: Ischemia and reperfusion (IR) injury remains a major cause of morbidity and mortality and multiple molecular and cellular pathways have been implicated in this injury. We determined whether acute inhibition of excessive mitochondrial fission at the onset of reperfusion improves mitochondrial dysfunction and cardiac contractility postmyocardial infarction in rats. METHODS AND RESULTS: We used a selective inhibitor of the fission machinery, P110, which we have recently designed. P110 treatment inhibited the interaction of fission proteins Fis1/Drp1, decreased mitochondrial fission, and improved bioenergetics in three different rat models of IR, including primary cardiomyocytes, ex vivo heart model, and an in vivo myocardial infarction model. Drp1 transiently bound to the mitochondria following IR injury and P110 treatment blocked this Drp1 mitochondrial association. Compared with control treatment, P110 (1 µmol/L) decreased infarct size by 28 ± 2% and increased adenosine triphosphate levels by 70+1% after IR relative to control IR in the ex vivo model. Intraperitoneal injection of P110 (0.5 mg/kg) at the onset of reperfusion in an in vivo model resulted in improved mitochondrial oxygen consumption by 68% when measured 3 weeks after ischemic injury, improved cardiac fractional shortening by 35%, reduced mitochondrial H2O2 uncoupling state by 70%, and improved overall mitochondrial functions. CONCLUSIONS: Together, we show that excessive mitochondrial fission at reperfusion contributes to long-term cardiac dysfunction in rats and that acute inhibition of excessive mitochondrial fission at the onset of reperfusion is sufficient to result in long-term benefits as evidenced by inhibiting cardiac dysfunction 3 weeks after acute myocardial infarction.