Mitochondrial ROS formation catalyzed by monoamine oxidase is causally related to inflammation, fibrosis and diastolic dysfunction in type 1 diabetic hearts

Cardiovascular disease is the leading cause of death among diabetic patients. Amongst various mechanisms proposed to contribute to the development of diabetic cardiomyopathy, oxidative stress has received significant experimental and clinical evaluation. However, large scale clinical trials using antioxidant therapies for the treatment of these pathological disorders have been ineffective. Collectively, these studies point towards a serious need to develop therapeutic strategies aimed at inhibiting specific sources of reactive oxygen species (ROS). In the present thesis, we investigated the role of monoamine oxidases (MAOs), outer mitochondrial enzymes that generate HβOβ, in oxidative stress and mitochondrial dysfunction in cardiomyocytes exposed to diabetic milieu and cardiac damage in type 1 diabetes (T1D) mice in vivo. Initially, we assessed ROS formation and mitochondrial function in primary cardiomyocytes treated with high glucose (HG) and/or interleukin-1β (IL-1β), a proinflammatory cytokine found to be elevated in diabetic patients. Cells exposed to these stimuli displayed an increase in ROS formation which was accompanied by mitochondrial dysfunction as documented by decreased mitochondrial membrane potential. MAO inhibitor pargyline completely prevented these alterations, suggesting that HG and IL-1β induce ROS formation and mitochondrial dysfunction in a MAO-dependent manner. Moreover, to study whether MAO activity is also involved in endoplasmic reticulum (ER)-mitochondria crosstalk and activation of unfolded protein response (UPR), we assessed markers of ER stress in this model. Interestingly, in adult cardiomyocytes, protein expression of activating transcription factor 4 (ATF4), growth arrest and DNA damage-inducible protein (GADDγ4), 78 kDa glucose-regulated protein (GRP78) and phosphorylation levels of IRE1α (inositolrequiring enzyme 1α) were significantly upregulated, marking the clear occurrence of ER stress. Pargyline administration abrogated these changes, suggesting that MAO is involved in HG and IL-1β induced UPR. Moreover, this suggests that, at least in this setting, MAOdependent ROS formation is upstream of ER stress, and play an important role in the crosstalk between mitochondria, inflammation and ER stress occurring in cardiomyocytes exposed to diabetic milieu. Given the involvement of MAO in HG and IL-1β induced cell damage, we investigated its role in cardiac dysfunction in streptozotocin (STZ)-induced T1D. We found that diastolic stiffness, an index of diastolic dysfunction, was significantly increased in STZ mice, whereas ejection fraction, an index of systolic function, remained unchanged. Moreover, markers of oxidative stress (4-hydroxynonenal) and UPR (ATF4 and GADDγ4) were significantly increased in STZ hearts as compared to controls. Importantly, STZ mice treated with MAO inhibitor pargyline displayed preserved diastolic function and absence of ER and oxidative stress. In agreement with previous reports showing that fibrosis is one of the major features of diabetic cardiomyopathy, we found that hearts from STZ-treated mice displayed increased collagen deposition. Interestingly, pargyline administration prevented this alteration, suggesting that MAO activity plays a crucial role in the progression of fibrosis in these animals. To understand whether MAO-mediated fibrosis was due to release of proinflammatory and pro-fibrotic factors from cardiac mast cells, we assessed mast cell degranulation. Indeed, mast cell degranulation increased by almost β-fold in STZ hearts as compared to control mice. MAO inhibition completely blocked the activation of mast cells in diabetic mice. These data indicate the novel role of these flavoenzymes in activating cardiac mast cell thereby leading to the remodeling of the extracellular matrix, fibrosis and ultimately, left ventricle (LV) dysfunction in T1D. Collectively, these results demonstrate that MAOs not only contribute to HG and inflammation induced mitochondrial ROS formation and dysfunction, but they also perturb ER function leading to the activation of UPR. Moreover, we showed that these flavoenzymes play a major role in the formation of a vicious cycle between oxidative stress and inflammation, which is likely the underlying cause of cardiac fibrosis and LV diastolic dysfunction in T1D mice. MAO inhibitors are clinically available and are being used for the treatment of several neurological and neurodegenerative diseases. Results from our study suggest that MAO inhibition could be a promising therapeutic strategy also for the treatment of cardiovascular complications in diabetes

Ribonucleotide synthesis by NME6 fuels mitochondrial gene expression

Replication of the mitochondrial genome and expression of the genes it encodes both depend on a sufficient supply of nucleotides to mitochondria. Accordingly, dysregulated nucleotide metabolism not only destabilises the mitochondrial genome, but also affects its transcription. Here, we report that a mitochondrial nucleoside diphosphate kinase, NME6, supplies mitochondria with pyrimidine ribonucleotides that are necessary for the transcription of mitochondrial genes. Loss of NME6 function leads to the depletion of mitochondrial transcripts, as well as destabilisation of the electron transport chain and impaired oxidative phosphorylation. These deficiencies are rescued by an exogenous supply of pyrimidine ribonucleosides. Moreover, NME6 is required for the maintenance of mitochondrial DNA when the access to cytosolic pyrimidine deoxyribonucleotides is limited. Our results therefore reveal an important role for ribonucleotide salvage in mitochondrial gene expression

Selective mitochondrial superoxide generation in vivo is cardioprotective through hormesis

Reactive oxygen species (ROS) have an equivocal role in myocardial ischaemia reperfusion injury. Within the cardiomyocyte, mitochondria are both a major source and target of ROS. We evaluate the effects of a selective, dose-dependent increase in mitochondrial ROS levels on cardiac physiology using the mitochondria-targeted redox cycler MitoParaquat (MitoPQ). Low levels of ROS decrease the susceptibility of neonatal rat ventricular myocytes (NRVMs) to anoxia/reoxygenation injury and also cause profound protection in an in vivo mouse model of ischaemia/reperfusion. However higher doses of MitoPQ resulted in a progressive alteration of intracellular [Ca2+] homeostasis and mitochondrial function in vitro, leading to dysfunction and death at high doses. Our data show that a primary increase in mitochondrial ROS can alter cellular function, and support a hormetic model in which low levels of ROS are cardioprotective while higher levels of ROS are cardiotoxic.The work is supported by an MRC Studentship to JFM and a Wellcome Trust Investigator award to RCH (110158/Z/15/Z), the Leducq Transatlantic Network of Excellence, and the University of Padova Strategico grant (FDL). Part of the study was funded by an MRC Project Grant to TK (MR/P000320/1). Michele Cariello is thanked for help with cyclic voltammetry

Monoamine oxidase-dependent endoplasmic reticulum-mitochondria dysfunction and mast cell degranulation lead to adverse cardiac remodeling in diabetes.

Monoamine oxidase (MAO) inhibitors ameliorate contractile function in diabetic animals, but the mechanisms remain unknown. Equally elusive is the interplay between the cardiomyocyte alterations induced by hyperglycemia and the accompanying inflammation. Here we show that exposure of primary cardiomyocytes to high glucose and pro-inflammatory stimuli leads to MAO-dependent increase in reactive oxygen species that causes permeability transition pore opening and mitochondrial dysfunction. These events occur upstream of endoplasmic reticulum (ER) stress and are abolished by the MAO inhibitor pargyline, highlighting the role of these flavoenzymes in the ER/mitochondria cross-talk. In vivo, streptozotocin administration to mice induced oxidative changes and ER stress in the heart, events that were abolished by pargyline. Moreover, MAO inhibition prevented both mast cell degranulation and altered collagen deposition, thereby normalizing diastolic function. Taken together, these results elucidate the mechanisms underlying MAO-induced damage in diabetic cardiomyopathy and provide novel evidence for the role of MAOs in inflammation and inter-organelle communication. MAO inhibitors may be considered as a therapeutic option for diabetic complications as well as for other disorders in which mast cell degranulation is a dominant phenomenon

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

The potential for ischemic preconditioning to reduce infarct size was first recognized more than 30 years ago. Despite extension of the concept to ischemic postconditioning and remote ischemic conditioning and literally thousands of experimental studies in various species and models which identified a multitude of signaling steps, so far there is only a single and very recent study, which has unequivocally translated cardioprotection to improved clinical outcome as the primary endpoint in patients. Many potential reasons for this disappointing lack of clinical translation of cardioprotection have been proposed, including lack of rigor and reproducibility in preclinical studies, and poor design and conduct of clinical trials. There is, however, universal agreement that robust preclinical data are a mandatory prerequisite to initiate a meaningful clinical trial. In this context, it is disconcerting that the CAESAR consortium (Consortium for preclinicAl assESsment of cARdioprotective therapies) in a highly standardized multi-center approach of preclinical studies identified only ischemic preconditioning, but not nitrite or sildenafil, when given as adjunct to reperfusion, to reduce infarct size. However, ischemic preconditioning—due to its very nature—can only be used in elective interventions, and not in acute myocardial infarction. Therefore, better strategies to identify robust and reproducible strategies of cardioprotection, which can subsequently be tested in clinical trials must be developed. We refer to the recent guidelines for experimental models of myocardial ischemia and infarction, and aim to provide now practical guidelines to ensure rigor and reproducibility in preclinical and clinical studies on cardioprotection. In line with the above guideline, we define rigor as standardized state-of-the-art design, conduct and reporting of a study, which is then a prerequisite for reproducibility, i.e. replication of results by another laboratory when performing exactly the same experiment

Mitochondrial ROS formation catalyzed by monoamine oxidase is causally related to inflammation, fibrosis and diastolic dysfunction in type 1 diabetic hearts

Cardiovascular disease is the leading cause of death among diabetic patients. Amongst various mechanisms proposed to contribute to the development of diabetic cardiomyopathy, oxidative stress has received significant experimental and clinical evaluation. However, large scale clinical trials using antioxidant therapies for the treatment of these pathological disorders have been ineffective. Collectively, these studies point towards a serious need to develop therapeutic strategies aimed at inhibiting specific sources of reactive oxygen species (ROS). In the present thesis, we investigated the role of monoamine oxidases (MAOs), outer mitochondrial enzymes that generate HβOβ, in oxidative stress and mitochondrial dysfunction in cardiomyocytes exposed to diabetic milieu and cardiac damage in type 1 diabetes (T1D) mice in vivo. Initially, we assessed ROS formation and mitochondrial function in primary cardiomyocytes treated with high glucose (HG) and/or interleukin-1β (IL-1β), a proinflammatory cytokine found to be elevated in diabetic patients. Cells exposed to these stimuli displayed an increase in ROS formation which was accompanied by mitochondrial dysfunction as documented by decreased mitochondrial membrane potential. MAO inhibitor pargyline completely prevented these alterations, suggesting that HG and IL-1β induce ROS formation and mitochondrial dysfunction in a MAO-dependent manner. Moreover, to study whether MAO activity is also involved in endoplasmic reticulum (ER)-mitochondria crosstalk and activation of unfolded protein response (UPR), we assessed markers of ER stress in this model. Interestingly, in adult cardiomyocytes, protein expression of activating transcription factor 4 (ATF4), growth arrest and DNA damage-inducible protein (GADDγ4), 78 kDa glucose-regulated protein (GRP78) and phosphorylation levels of IRE1α (inositolrequiring enzyme 1α) were significantly upregulated, marking the clear occurrence of ER stress. Pargyline administration abrogated these changes, suggesting that MAO is involved in HG and IL-1β induced UPR. Moreover, this suggests that, at least in this setting, MAOdependent ROS formation is upstream of ER stress, and play an important role in the crosstalk between mitochondria, inflammation and ER stress occurring in cardiomyocytes exposed to diabetic milieu. Given the involvement of MAO in HG and IL-1β induced cell damage, we investigated its role in cardiac dysfunction in streptozotocin (STZ)-induced T1D. We found that diastolic stiffness, an index of diastolic dysfunction, was significantly increased in STZ mice, whereas ejection fraction, an index of systolic function, remained unchanged. Moreover, markers of oxidative stress (4-hydroxynonenal) and UPR (ATF4 and GADDγ4) were significantly increased in STZ hearts as compared to controls. Importantly, STZ mice treated with MAO inhibitor pargyline displayed preserved diastolic function and absence of ER and oxidative stress. In agreement with previous reports showing that fibrosis is one of the major features of diabetic cardiomyopathy, we found that hearts from STZ-treated mice displayed increased collagen deposition. Interestingly, pargyline administration prevented this alteration, suggesting that MAO activity plays a crucial role in the progression of fibrosis in these animals. To understand whether MAO-mediated fibrosis was due to release of proinflammatory and pro-fibrotic factors from cardiac mast cells, we assessed mast cell degranulation. Indeed, mast cell degranulation increased by almost β-fold in STZ hearts as compared to control mice. MAO inhibition completely blocked the activation of mast cells in diabetic mice. These data indicate the novel role of these flavoenzymes in activating cardiac mast cell thereby leading to the remodeling of the extracellular matrix, fibrosis and ultimately, left ventricle (LV) dysfunction in T1D. Collectively, these results demonstrate that MAOs not only contribute to HG and inflammation induced mitochondrial ROS formation and dysfunction, but they also perturb ER function leading to the activation of UPR. Moreover, we showed that these flavoenzymes play a major role in the formation of a vicious cycle between oxidative stress and inflammation, which is likely the underlying cause of cardiac fibrosis and LV diastolic dysfunction in T1D mice. MAO inhibitors are clinically available and are being used for the treatment of several neurological and neurodegenerative diseases. Results from our study suggest that MAO inhibition could be a promising therapeutic strategy also for the treatment of cardiovascular complications in diabetes.Le malattie cardiovascolari sono le principali cause di morte tra i pazienti diabetici. Tra i vari meccanismi che contribuiscono allo sviluppo della cardiomiopatia diabetica, lo stress ossidativo ha ricevuto un’attenzione clinica e sperimentale significativa. Tuttavia, l’uso di terapie antiossidanti in studi clinici su larga scala è risultato inefficace nel trattamento di questi disturbi patologici. Nel complesso, questi studi indicano una reale necessità di sviluppare strategie terapeutiche volte a inibire specifiche fonti di specie reattive dell'ossigeno (ROS). Lo scopo di questa tesi è stato quello di valutare il ruolo delle monoammino ossidasi (MAO), enzimi mitocondriali localizzati nella membrana esterna, nello stress ossidativo e nella disfunzione mitocondriale in cardiomiociti esposti ad elevate concentrazione di glucosio (in vitro) ed il loro contributo in vivo nei danni cardiaci in un modello di T1D. Inizialmente, abbiamo valutato la formazione di ROS e la funzione mitocondriale in cardiomiociti primari trattati con alti livelli di glucosio (HG) e/o interleuchina-1β (IL-1β), una citochina pro-infiammatoria presente in livelli elevati in pazienti diabetici. Le cellule esposte a questi stimoli mostrano un aumento della formazione di ROS, accompagnata da disfunzione mitocondriale, come determinato dal minore potenziale di membrana mitocondriale. La pargilina, un inibitore MAO, previene completamente queste alterazioni, suggerendo che HG e IL-1β inducano la formazione di ROS e le disfunzione mitocondriale MAO-dipendente. Inoltre, per valutare se l’attività della MAO sia coinvolta nell’interazione tra i mitocondri ed il reticolo endoplasmatico (ER) e se possa determinare l’attivazione del’Unfolded Protein Response (UPR), in questo modello abbiamo misurato marcatori di stress dell’ER. È interessante notare come, in cardiomiociti adulti, l'espressione della proteina transcription factor 4 (ATF4), della growth arrest and DNA damage-inducible protein (GADD34), 78 kDa glucose-regulated protein (GRP78) e i livelli di fosforillazione di IRE1α (inositol-requiring enzyme 1α) siano significativamente elevati, il che dimostra la chiara presenza di stress del reticolo (ER). La somministrazione di pargilina previene e blocca queste alterazioni, suggerendo che MAO sia coinvolto nel processo di UPR indotto dalla combinazione di alto glucosio e IL-1β. Inoltre questi dati suggeriscono che, almeno in queste condizioni, la formazione di ROS MAO-dipendente è a monte dello stress del reticolo (ER) e svolge un ruolo importante nell’interazione tra mitocondri, infiammazione del reticolo in cardiomiociti esposti a condizioni simulanti il diabete. Dato il coinvolgimento della MAO nel danno cellulare causato da HG e IL-1β, abbiamo valutato il suo ruolo nella disfunzione cardiaca in modelli murini di diabete di tipo 1 (T1D) indotta da trattamento con streptozotocina (STZ). La rigidità diastolica, un indice di disfunzione diastolica, era significativamente aumentata nei topi STZ, mentre la frazione di eiezione, un indice di funzione sistolica, è rimasta invariata. Inoltre, i marcatori di stress ossidativo (4-idrossinonenale) e UPR (ATF4 e GADD34) sono risultati significativamente aumentati nei cuori STZ rispetto al controllo. È importante sottolineare come i topi diabetici trattati con la pargilina, inibitore specifico per MAO, abbiano mostrato una funzione diastolica preservata e l’assenza di stress ossidativo e dell’ ER. In accordo con studi precedenti dove si dimostra come la fibrosi è una delle caratteristiche principali nella cardiomiopatia diabetica, i cuori dei topi diabetici evidenziano una maggiore deposizione di collagene. È interessante notare che la somministrazione di pargilina ha impedito questa alterazione, suggerendo che l'attività MAO rivesta un ruolo cruciale nella progressione della fibrosi in questi animali. Al fine di determinare se la fibrosi MAO-mediata sia dovuta al rilascio di fattori pro-infiammatori e pro-fibrotici da mastociti cardiaci, abbiamo valutato la loro degranulazione. Abbiamo osservato che, la degranulazione dei mastociti è aumentata di quasi 2 volte nei cuori diabetici rispetto ai topi di controllo. L’inibizione MAO ha completamente bloccato l'attivazione dei mastociti nei topi diabetici. Questi dati indicano un ruolo completamente nuovo di questi flavoenzimi nell'attivare mastociti cardiaci alla base del rimodellamento della matrice extracellulare, nella fibrosi e in ultima analisi, nella disfunzione del ventricolo sinistro (LV) in T1D. Complessivamente, questi risultati dimostrano non solo come le MAO contribuisca alla formazione di ROS e alla disfunzione mitocondriale indotta da HG e dall’infiammazione, ma anche che queste specie reattive perturbano la funzione dell’ER e portano all’attivazione dell’UPR. Inoltre, abbiamo dimostrato come questi flavoenzimi svolgano un ruolo importante nella formazione di un circolo vizioso tra stress ossidativo e infiammazione, che è probabilmente la causa della fibrosi cardiaca e della disfunzione diastolica ventricolare sinistra nei topi diabetici. Gli inibitori MAO sono clinicamente disponibili e vengono utilizzati per il trattamento di diverse malattie neurologiche e neurodegenerative. I risultati del nostro studio suggeriscono come l'inibizione MAO potrebbe essere una strategia terapeutica promettente anche per il trattamento delle complicazioni cardiovascolari nel diabete

Antimicrobial peptides play a functional role in bumblebee anti-trypanosome defense

Bumblebees, amongst the most important of pollinators, are under enormous population pressures. One of these is disease. The bumblebee and its gut trypanosome Crithidia bombi are one of the fundamental models of ecological immunology. Although there is previous evidence of increased immune gene expression upon Crithidia infection, recent work has focussed on the bumblebee’s gut microbiota. Here, by knocking down gene expression using RNAi, we show for the first time that antimicrobial peptides (AMPs) have a functional role in anti-Crithidia defense

Mitochondrial Proteases: Multifaceted Regulators of Mitochondrial Plasticity

Mitochondria are essential metabolic hubs that dynamically adapt to physiological demands. More than 40 proteases residing in different compartments of mitochondria, termed mitoproteases, preserve mitochondrial proteostasis and are emerging as central regulators of mitochondrial plasticity. These multifaceted enzymes limit the accumulation of short-lived, regulatory proteins within mitochondria, modulate the activity of mitochondrial proteins by protein processing, and mediate the degradation of damaged proteins. Various signaling cascades coordinate the activity of mitoproteases to preserve mitochondrial homeostasis and ensure cell survival. Loss of mitoproteases severely impairs the functional integrity of mitochondria, is associated with aging, and causes pleiotropic diseases. Understanding the dual function of mitoproteases as regulatory and quality control enzymes will help unravel the role of mitochondrial plasticity in aging and disease

Supercritical antisolvent method for recrystallization of HMX

Supercritical antisolvent (SAS) method is an emerging technique for particle processing of high energetic materials. The study investigates the recrystallization of high energy material HMX (octahydro- 1,3,5,7-tetranitro-1,3,5,7-tetrazocine) using SAS method. The effect of pressure, solution flow rate, supercritical antisolvent flow rate and temperature on particle size and morphology of HMX crystals has been studied with acetone as solvent and supercritical carbon dioxide as antisolvent. Stable and desirable - polymorphic form of HMX could be obtained under certain process conditions and has been confirmed by FTIR spectroscopy. The experimental results show that - polymorph of HMX is of rhombohedral morphology with mean particle size of 13.7 μm, as confirmed by SEM and particle size analyzer respectively