THE ROLE OF THE MITOCHONDRIAL CALCIUM UNIPORTER (MCU) IN THE CARDIAC INJURY INDUCED BY ISCHEMIA AND REPERFUSION

Mitochondrial Ca2+ uptake has been suggested to contribute to the cardiac injury induced by ischemia reperfusion. This notion has been derived from studies using pharmacological approaches due to the lack of information in protein involved in mitochondrial Ca2+ uptake (Ferrari, Di Lisa et al. 1982). The recent identification of the molecular identity of the mitochondrial Ca2+ uniporter (MCU) (De Stefani, Raffaello et al. 2011) allows a genetic approaches. Based on the available notion MCU deletion could be protected against I/R injury , that should be exacerbated by MCU overexpression. The present result provide a more complex picture where by a model increase in mitochondrial Ca2+ elicits cardioprotection that is lost under condition of mitochondrial Ca2+ overload. Neonatal rat ventricular myocytes (NRVMs) overexpressing MCU by adenovirus infection showed a reduction in I/R-induced cell death as compared to wild type (wt) cells (41.82% ±8.37 vs 60.44% ±11.68, p<0.05). The in vitro evidence of cardioprotection was confirmed also ex vivo in perfused hearts overexpressing MCU by means of adenoassociated virus infection. Indeed, reperfusion after 40 min of global ischemia resulted in a significant decrease of lactate dehydrogenase release as compared to wt hearts (16.14 ±11.69 vs 67.01 ±0.07). This increased tolerance to I/R injury was associated with a large decrease in levels of reactive oxygen species (ROS) upon reperfusion. However, starting at 12 h after infection NRVMs displayed a slight increase in ROS levels associated with an increase in Akt phosphorylation (1.98± 0.06 fold) leading to the activation of this pro-survival kinase. Upstream of Akt, protein phosphatase 2A (PP2A) was more phosphorylated (2.8 ± 0.26 fold) resulting in its inactivation. Notably, Akt activation is abolished by antioxidants treatment. Overall, these findings suggest that a slight increase in mitochondrial Ca2+ induced by MCU overexpression triggers a protective response involving a mild oxidative stress that eventually stimulates the activity of survival pathways. The protection by MCU overexpression was abolished when a further increase in mitochondrial Ca2+ was induced by the co-expression of MICU1. This latter evidence confirms that mitochondrial Ca2+ overload is a determining factor in the loss of cardiac viability occurring during post ischemic reperfusion. Therefore, the balance between protection and injury appears to be modulated by levels of intramitochondrial Ca2+. In this respect, the results of this Thesis provide novel evidence that a mild increase in mitochondrial Ca2+ elicits cardioprotection by stimulating ROS formation. It is tempting to speculate that this mechanism is involved also in the protective effect against cardiac diseases induced by exercise

THE ROLE OF THE MITOCHONDRIAL CALCIUM UNIPORTER (MCU) IN THE CARDIAC INJURY INDUCED BY ISCHEMIA AND REPERFUSION

Mitochondrial Ca2+ uptake has been suggested to contribute to the cardiac injury induced by ischemia reperfusion. This notion has been derived from studies using pharmacological approaches due to the lack of information in protein involved in mitochondrial Ca2+ uptake (Ferrari, Di Lisa et al. 1982). The recent identification of the molecular identity of the mitochondrial Ca2+ uniporter (MCU) (De Stefani, Raffaello et al. 2011) allows a genetic approaches. Based on the available notion MCU deletion could be protected against I/R injury , that should be exacerbated by MCU overexpression. The present result provide a more complex picture where by a model increase in mitochondrial Ca2+ elicits cardioprotection that is lost under condition of mitochondrial Ca2+ overload. Neonatal rat ventricular myocytes (NRVMs) overexpressing MCU by adenovirus infection showed a reduction in I/R-induced cell death as compared to wild type (wt) cells (41.82% ±8.37 vs 60.44% ±11.68, p<0.05). The in vitro evidence of cardioprotection was confirmed also ex vivo in perfused hearts overexpressing MCU by means of adenoassociated virus infection. Indeed, reperfusion after 40 min of global ischemia resulted in a significant decrease of lactate dehydrogenase release as compared to wt hearts (16.14 ±11.69 vs 67.01 ±0.07). This increased tolerance to I/R injury was associated with a large decrease in levels of reactive oxygen species (ROS) upon reperfusion. However, starting at 12 h after infection NRVMs displayed a slight increase in ROS levels associated with an increase in Akt phosphorylation (1.98± 0.06 fold) leading to the activation of this pro-survival kinase. Upstream of Akt, protein phosphatase 2A (PP2A) was more phosphorylated (2.8 ± 0.26 fold) resulting in its inactivation. Notably, Akt activation is abolished by antioxidants treatment. Overall, these findings suggest that a slight increase in mitochondrial Ca2+ induced by MCU overexpression triggers a protective response involving a mild oxidative stress that eventually stimulates the activity of survival pathways. The protection by MCU overexpression was abolished when a further increase in mitochondrial Ca2+ was induced by the co-expression of MICU1. This latter evidence confirms that mitochondrial Ca2+ overload is a determining factor in the loss of cardiac viability occurring during post ischemic reperfusion. Therefore, the balance between protection and injury appears to be modulated by levels of intramitochondrial Ca2+. In this respect, the results of this Thesis provide novel evidence that a mild increase in mitochondrial Ca2+ elicits cardioprotection by stimulating ROS formation. It is tempting to speculate that this mechanism is involved also in the protective effect against cardiac diseases induced by exercise.L’uptake di Ca2+ mitocondriale contribuisce al danno cardiaco indotto da ischemia/riperfusione. Questo concetto è derivato da numerosi studi che hanno valutato il ruolo della proteina deputata all’uptake di Ca2+ mitocondriale servendosi di un approccio farmacologico. Tuttavia, la recente identificazione della struttura molecolare del canale responsabile dell’uptake di calcio definito MCU, ha reso possibile un approccio di tipo genetico, evitando i numerosi effetti collaterali degli inibitori farmacologici. Basandosi su i dati finora raccolti si presuppone che il silenziamento di MCU porti ad una riduzione del danno cardiaco in seguito ad I/R, e al contrario la sua sovraespressione ad un aumento del danno. Tuttavia i dati presentati in questa tesi mostrano un quadro più complesso in cui un moderato aumento del Ca2+ induce un effetto cardioprotettivo, che invece viene abrogato da un eccessivo carico di Ca2+ a livello mitocondriale. Cardiomiociti neonatali di ratto sovraesprimenti MCU tramite un infezione con adenovirus, mostrano una riduzione della mortalità sottoposti ad un protocollo di I/R (41.82%±8.37 vs 60.44%±11.68, p<0.05). L’evidenzia di questo effetto cardioprotettivo viene confermato anche da dati ottenuti ex vivo, in topi infettati con un virus adeno-associato di tipo 9 codificante per MCU-flag. Il cuore isolato sovraesprimente MCU sottoposto ad un protocollo di I/R in Langendorff mostra una riduzione della mortalità se comparato ad animali controllo (17.14±7.71 vs 30.16 ±10.35). Questa marcata riduzione della mortalità è accompagnata da una riduzione dello stress ossidativo in seguito all’evento post ischemico. Tuttavia, i cardiomiociti neonatali sovraesprimenti MCU mostrano un aumento dei ROS a livello basale, che correla con l’attivazione di Akt, chinasi coinvolta nei meccanismi di sopravvivenza cellulare. PP2A, fosfatasi coinvolta nella regolazione a monte di Akt, risulta essere più fosforilata quando MCU è sovraespresso, risultando perciò inattiva. Inoltre, l’attivazione di Akt viene abolita in seguito al trattamento con antiossidanti. Queste evidenze suggeriscono che un moderato aumento dell’uptake di Ca2+ mitocondriale indotto dalla sovraespressione di MCU sia responsabile dell’attivazione di un meccanismo di cardioprotezione che porta all’attivazione di meccanismi di sopravvivenza cellulare. Tuttavia, la cardioprotezione indotta dalla sola sovraespressione di MCU viene abrogata dalla co-espressione di MCU e MICU1, che determinano un massivo aumento di Ca2+ mitocondriale. Quest’ultima osservazione conferma che l’overload di Ca2+ mitocondriale è un fattore determinante nella mortalità indotta dal danno ischemico. Inoltre, appare evidente che il livello di Ca2+ mitocondriale sia il fattore determinante tra danno e protezione cardiaca. Questa tesi dimostra come un moderato aumento di Ca2+ mitocondriale possa determinare un effetto cardio-protettivo mediato da ROS. Inoltre, si potrebbe speculare che questo meccanismo di protezione rimandi all’effetto cardio-protettivo indotto dall’esercizio fisico

Hepatitis B virus PreS2-mutant large surface antigen activates store-operated calcium entry and promotes chromosome instability

[[abstract]]Hepatitis B virus (HBV) is a driver of hepatocellular carcinoma, and two viral products, X and large surface antigen (LHBS), are viral oncoproteins. During chronic viral infection, immune-escape mutants on the preS2 region of LHBS (preS2-LHBS) are gain-of-function mutations that are linked to preneoplastic ground glass hepatocytes (GGHs) and early disease onset of hepatocellular carcinoma. Here, we show that preS2-LHBS provoked calcium release from the endoplasmic reticulum (ER) and triggered stored-operated calcium entry (SOCE). The activation of SOCE increased ER and plasma membrane (PM) connections, which was linked by ER- resident stromal interaction molecule-1 (STIM1) protein and PM-resident calcium release- activated calcium modulator 1 (Orai1). Persistent activation of SOCE induced centrosome overduplication, aberrant multipolar division, chromosome aneuploidy, anchorage-independent growth, and xenograft tumorigenesis in hepatocytes expressing preS2- LHBS. Chemical inhibitions of SOCE machinery and silencing of STIM1 significantly reduced centrosome numbers, multipolar division, and xenograft tumorigenesis induced by preS2-LHBS. These results provide the first mechanistic link between calcium homeostasis and chromosome instability in hepatocytes carrying preS2-LHBS. Therefore, persistent activation of SOCE represents a novel pathological mechanism in HBV-mediated hepatocarcinogenesis

Live Cell Imaging in Microfluidic Device Proves Resistance to Oxygen/Glucose Deprivation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Analyses of cellular responses to fast oxygen dynamics are challenging and require ad hoc technological solutions, especially when decoupling from liquid media composition is required. In this work, we present a microfluidic device specifically designed for culture analyses with high resolution and magnification objectives, providing full optical access to the cell culture chamber. This feature allows fluorescence-based assays, photoactivated surface chemistry, and live cell imaging under tightly controlled pO₂ environments. The device has a simple design, accommodates three independent cell cultures, and can be employed by users with basic cell culture training in studies requiring fast oxygen dynamics, defined media composition, and in-line data acquisition with optical molecular probes. We apply this technology to produce an oxygen/glucose deprived (OGD) environment and analyze cell mortality in murine and human cardiac cultures. Neonatal rat ventricular cardiomyocytes show an OGD time-dependent sensitivity, resulting in a robust and reproducible 66 ± 5% death rate after 3 h of stress. Applying an equivalent stress to human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) provides direct experimental evidence for fetal-like OGD-resistant phenotype. Investigation on the nature of such phenotype exposed large glycogen deposits. We propose a culture strategy aimed at depleting these intracellular energy stores and concurrently activate positive regulation of aerobic metabolic molecular markers. The observed process, however, is not sufficient to induce an OGD-sensitive phenotype in hiPS-CMs, highlighting defective development of mature aerobic metabolism <i>in vitro</i>

Live Cell Imaging in Microfluidic Device Proves Resistance to Oxygen/Glucose Deprivation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Analyses of cellular responses to fast oxygen dynamics are challenging and require ad hoc technological solutions, especially when decoupling from liquid media composition is required. In this work, we present a microfluidic device specifically designed for culture analyses with high resolution and magnification objectives, providing full optical access to the cell culture chamber. This feature allows fluorescence-based assays, photoactivated surface chemistry, and live cell imaging under tightly controlled pO₂ environments. The device has a simple design, accommodates three independent cell cultures, and can be employed by users with basic cell culture training in studies requiring fast oxygen dynamics, defined media composition, and in-line data acquisition with optical molecular probes. We apply this technology to produce an oxygen/glucose deprived (OGD) environment and analyze cell mortality in murine and human cardiac cultures. Neonatal rat ventricular cardiomyocytes show an OGD time-dependent sensitivity, resulting in a robust and reproducible 66 ± 5% death rate after 3 h of stress. Applying an equivalent stress to human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) provides direct experimental evidence for fetal-like OGD-resistant phenotype. Investigation on the nature of such phenotype exposed large glycogen deposits. We propose a culture strategy aimed at depleting these intracellular energy stores and concurrently activate positive regulation of aerobic metabolic molecular markers. The observed process, however, is not sufficient to induce an OGD-sensitive phenotype in hiPS-CMs, highlighting defective development of mature aerobic metabolism <i>in vitro</i>

Live Cell Imaging in Microfluidic Device Proves Resistance to Oxygen/Glucose Deprivation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Analyses of cellular responses to fast oxygen dynamics are challenging and require ad hoc technological solutions, especially when decoupling from liquid media composition is required. In this work, we present a microfluidic device specifically designed for culture analyses with high resolution and magnification objectives, providing full optical access to the cell culture chamber. This feature allows fluorescence-based assays, photoactivated surface chemistry, and live cell imaging under tightly controlled pO₂ environments. The device has a simple design, accommodates three independent cell cultures, and can be employed by users with basic cell culture training in studies requiring fast oxygen dynamics, defined media composition, and in-line data acquisition with optical molecular probes. We apply this technology to produce an oxygen/glucose deprived (OGD) environment and analyze cell mortality in murine and human cardiac cultures. Neonatal rat ventricular cardiomyocytes show an OGD time-dependent sensitivity, resulting in a robust and reproducible 66 ± 5% death rate after 3 h of stress. Applying an equivalent stress to human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) provides direct experimental evidence for fetal-like OGD-resistant phenotype. Investigation on the nature of such phenotype exposed large glycogen deposits. We propose a culture strategy aimed at depleting these intracellular energy stores and concurrently activate positive regulation of aerobic metabolic molecular markers. The observed process, however, is not sufficient to induce an OGD-sensitive phenotype in hiPS-CMs, highlighting defective development of mature aerobic metabolism <i>in vitro</i>