Dissertations / Theses on the topic 'Unipore calcique mitochondrial'

La recherche de molécules ou d'outils thérapeutiques innovants est au cœur des préoccupations scientifiques. Au niveau cardiaque, le développement de pathologies comme l'ischémie-reperfusion et la cardiomyopathie diabétique a des effets délétères sur la structure et la fonction du cœur. Dans notre étude, nous avons testé deux substances prometteuses : le kaempférol et les vésicules extracellulaires portant le morphogène Sonic Hedgehog (VESHH).Le kaempférol, un polyphénol présent dans les fruits et légumes, possède des propriétés anti-inflammatoires, antioxydantes et anti-apoptotiques. Il a montré des effets anti-arythmiques, notamment dans la cardiomyopathie diabétique, en activant le complexe MCU qui régule l'entrée du calcium dans la mitochondrie. Cependant, les effets précis du kaempférol sur le complexe MCU en fonction de son assemblage restent indéterminés. D’une part, nous avons démontré que le calcium modifie la conformation des sous-unités MICU1, permettant au kaempférol de se fixer et de réarranger ces sous-unités. D’autre part, les effets du kaempférol sur les propriétés biophysiques du complexe MCU sont régis par le ratio MICU1/MCU. En effet, le kaempférol n’induit aucune modification des courants unitaires MCUC sur un fort ratio MICU1/MCU, tel que trouvé dans des particules sub-mitochondriales de cœur. En revanche, l’effet activateur du kaempférol sur les propriétés biophysiques du MCUC est retrouvé avec un faible ratio MICU1/MCU dans des SMP de foie dès 1 µM de calcium. Ainsi, l'utilisation du kaempférol, afin d’activer l’absorption de calcium mitochondrial par le MCUC, doit être adaptée en fonction de la pathologie cardiaque et de ses dysfonctions métaboliques, mais aussi du ratio MICU1/MCU. De plus, la diversité d'effets du kaempférol rend son utilisation clinique complexe et incertaine selon les pathologies.Les VESHH ont été testées dans un modèle d'ischémie-reperfusion de 28 jours chez le porc. Produites à partir de lymphocytes T, ces VESHH n'ont pas montré d'effets bénéfiques sur les lésions cardiaques, quel que soit le temps de reperfusion, en raison de leur inactivité. Si les résultats des VESHH se révèlent peu concluants dans cette étude, des travaux antérieurs mettent en avant leur potentiel thérapeutique dans l'ischémie-reperfusion cardiaque au bout de 24 heures de reperfusion chez le gros animal, bien plus prometteur lorsqu'elles sont biologiquement actives, en diminuant les marqueurs de souffrance myocardique, la taille de la lésion infarcie et même les arythmies ventriculaires lors de la reperfusion.En résumé, bien que le kaempférol et les VESHH soient des approches thérapeutiques différentes, ciblent des mécanismes différents et des pathologies cardiaques distinctes, elles apparaissent comme des molécules prometteuses. Des recherches supplémentaires seront nécessaires pour approfondir leur potentiel en tant que traitements thérapeutiques
The search for innovative molecules or therapeutic tools is at the heart of scientific concerns. In cardiology, the development of pathologies such as ischemia-reperfusion and diabetic cardiomyopathy has deleterious effects on the structure and function of the heart. In our study, we tested two promising substances: kaempferol and extracellular vesicles carrying the Sonic Hedgehog morphogen (EVSHH).Kaempferol, a polyphenol found in fruits and vegetables, has anti-inflammatory, antioxidant, and anti-apoptotic properties. It has shown anti-arrhythmic effects, particularly in diabetic cardiomyopathy, by activating the MCU complex that regulates calcium entry into the mitochondria. However, the precise effects of kaempferol on the MCU complex depending on its assembly remain undetermined. On one hand, we demonstrated that calcium modifies the conformation of MICU1 subunits, allowing kaempferol to bind and rearrange these subunits. On the other hand, the effects of kaempferol on the biophysical properties of the MCU complex are governed by the MICU1/MCU ratio. Indeed, kaempferol does not induce any modification of MCUC unitary currents with a high MICU1/MCU ratio, as found in heart sub-mitochondrial particles. Conversely, the activating effect of kaempferol on the biophysical properties of MCUC is observed with a low MICU1/MCU ratio in liver SMPs from 1 µM calcium. Thus, the use of kaempferol to activate mitochondrial calcium uptake by MCUC must be adapted according to the cardiac pathology and its metabolic dysfunctions, as well as the MICU1/MCU ratio. Moreover, the diversity of kaempferol's effects makes its clinical use complex and uncertain depending on the targeted pathologies.EVSHH were tested in a 28-day ischemia-reperfusion model in pigs. Produced from T lymphocytes, EVSHH did not show beneficial effects on cardiac damage, regardless of the reperfusion time, due to their inactivity. While the results of EVSHH are inconclusive in this study, previous work highlights their therapeutic potential in cardiac ischemia-reperfusion after 24 hours of reperfusion in large animals, being much more promising when they are biologically active, by reducing markers of myocardial injury, infarct size, and even ventricular arrhythmias during reperfusion.In summary, although kaempferol and EVSHH are different therapeutic approaches targeting different mechanisms and distinct cardiac pathologies, they appear as promising molecules. Further research will be necessary to deepen their potential as therapeutic treatments

Biochemistry
Ph.D.
Mitochondrial calcium (Ca2+) uptake has been studied for over five decades, with crucial insights into its underlying mechanisms enabled by development of the chemi-osmotic hypothesis and appreciation of the considerable voltage present across the inner mitochondrial membrane (ΔΨm) generated by proton pumping by the respiratory chain (Carafoli, 1987; Nicholls, 2005). However, the molecules that regulate mitochondrial Ca2+ uptake have only recently been identified (Jiang et. al., 2009; Perocchi et. al., 2010) and further work was needed to clarify how these molecules regulate mitochondrial Ca2+ uptake. Leucine Zipper EF hand containing Transmembrane Protein 1 (LETM1) acts as a regulator of mitochondrial Ca2+ uptake distinct from the mitochondrial Ca2+ uniporter (MCU) pathway (Jiang et. al., 2009). However, a controversy exists regarding the function of LETM1 (Nowikovsky et. al., 2004). Therefore, I asked if LETM1 played a role in mitochondrial Ca2+ uptake and if LETM1 regulated cellular bioenergetics and basal autophagy. To further characterize mitochondrial calcium uptake, we asked how Mitochondrial Calcium Uptake 1 (MICU1) regulates MCU activity by quantifying basal mitochondrial Ca2+ and MCU uptake rates in MICU1 ablated cells. The following work characterizes the molecules that regulate mitochondrial Ca2+ uptake and their mechanistic function on decoding calcium signals. Since LETM1 is the Ca2+/H+ antiporter, I hypothesize that alterations in LETM1 expression and activity will decrease mitochondrial Ca2+ uptake and will result in impaired mitochondrial bioenergetics. As a regulator of free intracellular Ca2+, mitochondrial Ca2+ uptake and the orchestra of its regulatory molecules have been implicated in many human diseases. Mitochondria act both upstream by regulating cytosolic Ca2+ concentration and as downstream effectors that respond to Ca2+ signals. Recently, LETM1 was proposed as a mitochondrial Ca2+/H+ antiporter (Jiang et. al., 2009); however characterization of the functional role of LETM1-mediated Ca2+ transfer remained unstudied. Therefore the specific aims of this project were to determine how LETM1 regulates Ca2+ homeostasis and bioenergetics under physiological settings. Secondly, this project aimed to characterize how LETM1-dependent Ca2+ signaling regulates ROS production and autophagy. The data presented here confirmed that LETM1 knockdown significantly impairs mitochondrial Ca2+ uptake. Furthermore, in-depth approaches including either deletion of EF-hand or mutation of critical EF-hand residues (D676A D688KLETM1) impaired histamine (GPCR agonist)-induced mitochondrial Ca2+ uptake. Knockdown of LETM1 resulted in bioenergetic collapse and promoted LC3-positive multilamellar vesicle formation, indicative of autophagy induction. Interestingly, knockdown of LETM1 significantly reduced complex IV but not complex I and complex II-mediated oxygen consumption rate (OCR). In contrast, cellular NADH and mitochondrial membrane potential (ΔΨm) were unaltered in both control and LETM1 knockdown cells. LETM1 has been implicated in formation of the supercomplexes of the electron transport chain (Tamai et. al., 2008). In support, these studies show that LETM1 knockdown results in increased reactive oxygen species (ROS) production. These results for the first time demonstrate that LETM1 controls cellular bioenergetics through regulation of mitochondrial Ca2+ and ROS. MICU1 was identified as an essential regulator of the mitochondrial Ca2+ uniporter (Perocchi et. al., 2010). Therefore, this project specifically aimed to determine how MICU1 regulates the mitochondrial Ca2+ uniporter. Interestingly, the data presented here suggest that MICU1 is not necessary for uniporter activity. Instead, loss of MICU1 caused mitochondria to constitutively load Ca2+ at rest which resulted in a host of cellular phenotypes. This result led to further questions on how MICU1 knockdown affects cellular bioenergetics and if MICU1 is essential for cell survival under stress. MICU1 ablation influenced pyruvate dehydrogenase activity and ROS production. Subsequent investigations demonstrated that increased basal ROS left cells poised to ceramide-induced cell death thereby suggesting the role of MICU1 in cell survival. Collectively, the data presented here show that MICU1 is necessary to control constitutive mitochondrial Ca2+ uptake during rest. This work demonstrates that LETM1 regulates a distinct mode of mitochondrial Ca2+ uptake pathway whereas MICU1 controls mitochondrial Ca2+ uniporter activity. Further studies are required to uncover the potential role of these two mitochondrial-resident Ca2+ regulators in health and disease.
Temple University--Theses

During neuronal activity mitochondria alter cytosolic Ca2+ signaling by buffering then releasing Ca2+ in the cytosol. This calcium transport by mitochondria affects the amplitude, duration, and spacial profile of the Ca2+ signal in the cytosol of neurons. This buffering by mitochondria has been shown to affect a variety of neuronal functions including: neurotransmission, gene expression, cell excitability, and cell death. Recently, researchers discovered that the protein CCDC109A (mitochondrial Ca2+ uniporter) was the protein responsible for mitochondrial Ca2+ uptake. Using a genetic knockout (KO) mouse model for the mitochondrial Ca2+ uniporter (MCU) my research investigated the role of MCU in neuronal function. In cultured central and peripheral neurons, MCU-KO significantly reduced mitochondrial Ca2+ uptake while significantly increasing the amplitude of the cytosolic Ca2+ signal amplitude. Behaviorally, MCU-KO mice show a small but significant impairment in memory tasks: fear conditioning and Barnes maze. Using a maximal electroshock seizure threshold model of in vivo seizure activity my research found that MCU-KO significantly increases the threshold for maximal seizure activity in mice and significantly reduces seizure severity. In addition to mitochondrial Ca2+ uptake, my research also investigated the mechanisms involved in mitochondrial Ca2+ extrusion. The protein SLC8B1 (SLC24A6, NCLX) is the putative transporter responsible for the Na+/Ca2+ exchange, mitochondrial calcium extrusion. Using genetic NCLX-KO mice, our research found that in neurons NCLX contributes to cytosolic Ca2+ extrusion, but does seem to directly affect mitochondrial Ca2+ extrusion.

By buffering cytosolic calcium, mitochondria can shape the magnitude and duration of intracellular calcium transients, which in turn govern key physiological events. Although controlled uptake of calcium into the matrix influences the rate of ATP production, excess calcium within the matrix triggers non-specific permeabilization of the mitochondrial inner membrane, resulting in cell death. Despite its importance in cellular physiology, the molecular identity of the mitochondrial calcium uniporter remained a mystery for nearly five decades. Recently, an approach inspired by comparative genomics was used to identify two proteins required for high-capacity mitochondrial calcium uptake. These include MICU1, an EF-hand protein that may function as a regulatory component by sensing calcium, and MCU, the channel-forming subunit of the uniporter. In this work, I explore two distinct areas within the growing field of molecular mitochondrial calcium biology. First, I discuss the identification of a new protein, MICU1-paralog EFHA1, and present data that implicates it in mitochondrial calcium uptake. Subsequently, I describe efforts to establish an in vitro system to characterize the channel activity of MCU, including my contribution to the development of a liposome-based assay for calcium transport and preliminary work aimed at reconstituting MCU transport activity in proteoliposomes.

In a wide variety of cell types, cytosolic Ca2+ transients, generated by physiological stimuli, elicit large increases in the [Ca2+] of the mitochondrial matrix, which in turn stimulate the Ca2+-sensitive dehydrogenases of the Krebs cycle. Rapid uptake is favored by the close proximity with the major Ca2+ store of the cell, namely the endoplasmic/sarcoplasmic reticulum (ER/SR), and thus by the exposure to high [Ca2+] microdomains. In addition, mitochondrial Ca2+ could contribute to the cellular homeostasis thanks to the existence of a sophisticated machinery, that allows this organelle to rapidly change its Ca2+ concentration (Rizzuto et al., 2012). This general picture is also apparent in skeletal muscle during contraction whereby agonist stimulation induces high amplitude mitochondrial Ca2+ increases in vivo (Rudolf et al., 2004), thus acting as buffers of the cytosolic [Ca2+] increase. Finally, mitochondrial Ca2+ stimulates aerobic metabolism and ATP production, that are essential for muscle activity. Indeed, mitochondria are the major source of ATP in oxidative fibres. However, excessive Ca2+ accumulation in mitochondria, a condition known as mitochondrial Ca2+ overload, can trigger cell death. The recent molecular identification of the Mitochondrial Calcium Uniporter (MCU), the highly selective channel responsible for Ca2+ entry into mitochondria, allows the detailed investigation of its role in different aspects of skeletal muscle biology (De Stefani et al., 2011; Baughman et al., 2011). The major goal of my PhD project was to address the role of mitochondrial Ca2+ in skeletal muscle homeostasis. For this purpose, we firstly investigated in vivo the effects of mitochondrial Ca2+ homeostasis in skeletal muscle function by overexpressing or silencing MCU by means of AAV vectors. We demonstrated that the modulation of MCU protein controls skeletal muscle size during both post-natal growth and adulthood. In detail, we observed an increase in fibre size in MCU-infected muscles. Conversely, MCU-silenced muscles displayed an atrophic phenotype. These striking phenomenon impinges on two major hypertrophic pathways, i.e. PGC-1α4 and IGF1-AKT. We thus explored two potential different mechanisms that could account for the MCU-dependent control of anabolic pathways, i) the activation of a mitochondria-to-nucleus signaling route, ii) the regulation of metabolites as signaling molecules. Regarding the mitochondria-to-nucleus route, we carried out a study on the PGC-1α4 promoter activity, and we demonstrated that mitochondrial Ca2+ controls the promoter activity of PGC-1α4. Concerning the involvement of cellular metabolism, we carried out steady-state metabolomics analyses of MCU-overexpressing and MCU-silencing muscles. We discovered a marked metabolic reprogramming in silenced muscles, including a clear shift from glucose metabolism toward preferential fatty acid β-oxidation. Next, we generated a skeletal muscle specific mcu knockout mouse (mlc1f-Cre-mcu-/-), by crossing a mcu fl/fl mouse with a line expressing the Cre recombinase under the control of the myosin light chain 1f (mlc1f) promoter. We observed marginal difference in fibre size of mlc1f-Cre-mcu-/- skeletal muscles. However, when these mice were exercised on a treadmill using different training protocols, an impaired running capacity of mlc1f-Cre-mcu-/- became evident, indicating that mitochondrial Ca2+ accumulation is required to guarantee skeletal muscle performance. Finally, it is well-established that Ca2+ plays a pivotal role in autophagy regulation. Thus, we decided to investigate this process in MCU-overexpressing and MCU-silencing muscles. We demonstrated that mitochondrial Ca2+ uptake modulation controls mitophagy without affecting bulk autophagy. Taken together, these data indicate that mitochondrial Ca2+ uptake plays a pivotal role in the control of skeletal muscle trophism. Further investigations of MCU-dependent effects on skeletal muscle homeostasis represent an important task for the future. Indeed, this research will provide new possible targets for clinical intervention in all diseases characterized by muscle loss, such as dystrophies, cancer cachexia and aging.
In diversi tipi cellulari, i transienti di Ca2+ citosolico, generati da stimoli fisiologici, provocano ampi aumenti della concentrazione di Ca2+ nella matrice mitocondriale, che, a loro volta, stimolano le deidrogenasi Ca2+-sensibili del ciclo di Krebs. Questo rapido accumulo è favorito dalla vicinanza al principale deposito di Ca2+ della cellula, il reticolo endo/sarcoplasmatico (RE/RS), e di conseguenza dalla generazione di microdomini ad elevata concentrazione di Ca2+. Inoltre, il Ca2+ mitocondriale contribuisce all’omeostasi cellulare grazie all’esistenza di un complesso macchinario che permette a questo organello di accumulare rapidamente grandi quantità di Ca2+ (Rizzuto et al., 2012). Questo situazione è presente anche nel muscolo scheletrico, in cui la stimolazione che genera contrazione induce ampi transienti di Ca2+ mitocondriale in vivo (Rudolf et al., 2004), che sono in grado di tamponare gli aumenti della concentrazione di Ca2+ citosolica. Infine, il Ca2+ mitocondriale stimola il metabolismo aerobico e la produzione di ATP, che sono essenziali per l’attività muscolare. Infatti, i mitocondri rappresentano la principale fonte di ATP nelle fibre ossidative. Tuttavia, un accumulo eccessivo di Ca2+ nei mitocondri può anche portare a morte cellulare. La recente scoperta dell’identità molecolare del Mitochondrial Calcium Uniporter (MCU), il canale altamente selettivo responsabile dell’entrata di Ca2+ nei mitocondri, permette lo studio dettagliato del suo ruolo nei diversi aspetti della biologia del muscolo scheletrico (Baughman et al., 2011; De Stefani et al., 2011). L’obiettivo principale del mio progetto di tesi è stato quello di scoprire il ruolo del Ca2+ mitocondriale nell’omeostasi del muscolo scheletrico. Per fare questo, per prima cosa abbiamo indagato in vivo come le funzioni muscolari vengono controllate dall’omeostasi mitocondriale del Ca2+ attraverso la sovraespressione o il silenziamento di MCU. Abbiamo dimostrato che la modulazione di MCU controlla la dimensione del muscolo scheletrico sia durante la crescita post-natale che nell’età adulta. In particolare, abbiamo osservato un aumento nella dimensione delle fibre nei muscoli infettati con MCU. Al contrario, i muscoli in cui MCU è stato silenziato risultano atrofici. Questo straordinario fenomeno dipende dal coinvolgimento delle due principali vie di segnalazione che mediano l’ipertrofia, ovvero PGC-1α4 e IGF1-AKT. Di conseguenza, abbiamo studiato due diversi meccanismi potenzialmente in grado di spiegare il controllo delle vie anaboliche dipendente da MCU, i) l’attivazione di una comunicazione diretta fra mitocondrio e nucleo, ii) l’azione di metaboliti come segnali. Per quanto riguarda la comunicazione mitocondrio-nucleo, abbiamo studiato l’attività del promotore di PGC-1α4, dimostrando che il Ca2+ mitocondriale la controlla. Invece, nel contesto dei metaboliti come molecole segnale, abbiamo svolto un’analisi metabolomica di muscoli in cui MCU è stato sovraespresso o silenziato. Abbiamo rilevato un notevole rimodellamento della rete metabolica nei muscoli silenziati, compresa una chiara deviazione dal metabolismo del glucosio verso la preferenziale ossidazione degli acidi grassi. In seguito, abbiamo generato un modello murino privo di mcu esclusivamente nel muscolo scheletrico (mlc1f-Cre-mcu-/-), incrociando un topo mcu fl/fl con una linea che esprime la Cre ricombinasi sotto il controllo del promotore per la catena leggera della miosina 1f (mlc1f). Abbiamo osservato differenze marginali per quanto riguarda la dimensione delle fibre muscolari di questo modello. Tuttavia, abbiamo poi sottoposto questi topi ad esercizio fisico, attraverso diversi protocolli di corsa su tapis roulant. In queste condizioni, è stata evidenziata una compromessa capacità di corsa, indicando che l’accumulo di Ca2+ mitocondriale è richiesto per garantire performance muscolari ottimali. Infine, è ampiamente riconosciuto che il Ca2+ giochi un ruolo fondamentale nella regolazione dell’autofagia. Abbiamo quindi deciso di studiare questo processo in muscoli in cui MCU è stato sovraespresso o silenziato. Abbiamo dimostrato che i segnali Ca2+ mitocondriali controllano selettivamente la via autofagica che degrada i mitocondri disfunzionali, la mitofagia. In conclusione, questi dati indicano che l’accumulo mitocondriale di Ca2+ controlla il trofismo del muscolo scheletrico. In futuro saranno necessari ulteriori studi per caratterizzare meglio gli effetti di MCU sull’omeostasi del muscolo scheletrico. Questo studio fornirà nuovi potenziali bersagli che sarà possibile utilizzare in clinica, in tutte quelle patologie caratterizzate dalla perdita di massa muscolare, come ad esempio le distrofie, la cachessia neoplastica e l’invecchiamento.

Mitochondria are cellular organelles that play a key role in several physiological processes, including cell proliferation, differentiation, cell death and the regulation of cellular calcium (Ca2+) homeostasis. Increases in mitochondrial Ca2+ activate several dehydrogenases and carriers, inducing enhance in the respiratory rate, H+ extrusion, and ATP production necessary for the correct energy state of the cell. The mitochondrial Ca2+ uptake and release mechanisms are based on the utilization of gated channels for Ca2+ uptake and exchangers for release that are dependent upon the negative mitochondrial membrane potential, which represents the driving force for Ca2+ accumulation in the mitochondrial matrix. In this thesis, the attention was focused on two mechanisms in particular, the mitochondrial Ca2+ influx system by the activity of Mitochondrial Calcium Uniporter (MCU) complex, and the high-conductance channel mitochondrial Permeability Transition Pore (mPTP), responsible for a state of non-selective permeability of the inner mitochondrial membrane (IMM); its opening in non-physiological conditions leads to Ca2+ release from mitochondria and triggers cell death mechanisms. Thus the maintenance of the mitochondrial Ca2+ homeostasis is essential for a proper balance between cell life or death. In particular it will be discussed the possible involvement of MCU in the cell cycle, as the Ca2+ accumulation by MCU is important for the regulation of cell life and energy production. It will be shown that MCU is mainly expressed in specific phases of the cell cycle and this expression positive correlates with the mitochondrial membrane potential. MCU overexpression instead does not alter cell cycle phases. It will also described the role of the c subunit of Fo ATP synthase in mitochondrial permeability transition (MPT) and it will be demonstrated to be a critical component of the mPTP complex. Finally it will be discussed the role of mPTP in mitochondrial Ca2+ efflux and it will be shown that it is a dispensable element for mitochondrial Ca2+ efflux in non-pathological conditions.

Biomedical Sciences
Ph.D.
Mitochondrial Calcium (mCa2+) overload is a central event in myocardial-ischemia reperfusion (IR) injury that leads to metabolic derangement as well as activation of the mitochondrial permeability transition pore (mPTP). mPTP activation results in necrosis and loss of cardiomyocytes which results in acute death in some individuals while survivors are prone to developing heart failure and are predisposed to recurrent infarction events. mCa2+ has also long been known to activate cellular bioenergetics implicating mCa2+ in the highly metabolically demanding state of cardiac contractility. The mitochondrial calcium uniporter channel (mtCU) is a multi-subunit complex that resides in the inner mitochondrial membrane and is required for mitochondrial Ca2+ (mCa2+) uptake. Mitochondrial Calcium Uniporter B (MCUB, CCDC109B gene), a recently identified paralog of MCU, is reported to negatively regulate mCa2+ uptake; however, its precise regulation of uniporter function and contribution to cardiac physiology remain unresolved. Size exclusion chromatography of mitochondria isolated from ventricular tissue revealed MCUB was undetectable in the high-molecular weight (MW) fraction of sham animals (~700kD, size of functional mtCU), but 24 hours following myocardial ischemia-reperfusion injury (IR) MCUB was clearly observed in the high-MW fraction. To investigate how MCUB contributes to mtCU regulation we created a stable MCUB-/- HeLa cell line using CRISPR-Cas9n. MCUB deletion increased histamine-mediated mCa2+ transient amplitude by ~50% versus Wild-Type (WT) controls (mito-R-GECO1). Further, MCUB deletion increased mtCU capacitance (patch-clamp) and rate of [mCa2+] uptake. Fast protein liquid chromatography (FPLC) fractionation of the mtCU revealed that loss of MCUB increased MCU incorporation into the high-MW complex suggesting stoichiometric replacement and overall increase in functional mtCU complexes. Next, we generated a cardiac-specific, tamoxifen-inducible MCUB mouse model (CAG-CAT-MCUB x MCM; MCUB-Tg) to examine how the MCUB/MCU ratio regulates mtCU function and contributes to cardiac physiology. MCUB-Tg mice were infected with AAV9-mitycam (genetic mCa2+ reporter) and adult cardiomyocytes were isolated to record [mCa2+] transients during pacing using live cell imaging. Increasing the MCUB/MCU ratio decreased [mCa2+] peak amplitude by ~30% and significantly reduced the [mCa2+] uptake rate. FPLC assessment revealed MCUB was undetectable in the high-MW fraction of MerCreMer controls, but enriched in MCUB-Tg hearts. MCUB incorporation into the mtCU decreased the overall size of the uniporter and reduced the presence of channel gatekeepers, MICU1/2. Immunoprecipitations suggest that MCUB directly interacts with MCU but does not bind MICU1/2. These results suggest that MCUB replaces MCU in the mtCU and thereby modulates the association of MICU1/2 to regulate channel gating. Cardiomyocytes isolated from MCUB-Tg hearts displayed decreased maximal respiration and reserve capacity, which correlated with a severe impairment in isoproterenol-induced contractile reserve (LV invasive hemodynamics). MCUB-Tg cardiac mitochondria were resistant to Ca2+-induced mitochondrial swelling suggesting MCUB limits mitochondrial permeability transition. Further, MCUB-Tg mice subjected to in vivo myocardial IR revealed a ~50% decrease in infarct size per area-at-risk suggesting increased MCUB expression prevents mCa2+ overload and limits cell death. These data suggest that MCUB regulation of the mtCU is an endogenous compensatory mechanism to decrease mCa2+ overload during ischemic injury, but this expression is maladaptive to cardiac energetic responsiveness and contractility.
Temple University--Theses

Mitochondrial Ca2+ buffering is an important physiological modulator of neuronal signaling and bioenergetics, but this propensity toward Ca2+ regulation proves pathological during excitotoxic insult. Specifically, excessive mitochondrial Ca2+ uptake is a key component of glutamate toxicity within the penumbra surrounding the ischemic core following stroke. This mitochondrial toxicity and Ca2+ dyshomeostasis may be visualized in real time as delayed calcium deregulation (DCD). DCD is a predictor of neuronal, excitoxic death, and is composed of three phases: 1) an initial response; 2) a latent period of elevated, but stable cytosolic Ca2+; and 3) failure of mitochondrial Ca2+ retention, termed deregulation. The duration of the latent period is an index of neuronal resistance. Mitochondria are dynamic organelles that rapidly and reversibly undergo fission and fusion (MFF). MFF is tightly regulated by the phosphoregulation of fission inducing Drp1 at serine 656. Drp1-S656 phosphorelation is mediated by PKA/AKAP1, and it is dephosphorylated by PP2A/Bβ2. Phosphorylation of Drp1-S656 inactivates this contractile GTPase resulting in inhibition of mitochondrial fission and a shift toward elongated mitochondria. This PKA/AKAP1 dependent Drp1-S656 phosphorylation has proven to be neuroprotective. Likewise, attenuation of PP2A/Bβ2 signaling enhances neuronal survival during ischemia and excitotoxic insult. Based on the mitochondrial buffering role in excitotoxicity and MFF modulation of neuronal survival, we began investigating the role of Ca2+ buffering as a function of MFF during glutamate toxicity. Noted above, resistance to excitoticity is visualized by the duration of the DCD latent period. Overexpression of AKAP1 in cultured hippocampal neurons greatly prolonged DCD latency in a PKA dependent manner, while Bβ2 ablation prolonged DCD latency by hours. Pharmacological modulation of PKA required PDE4 inhibition to reproduce the AKAP1 observations. Preliminary experiments studying the effect of Bβ2 overexpression on matrix Ca2+ load suggests possible mechanism of MFF regulated of matrix Ca2+ accumulation. Using mtPericam DRG neurons as a model system for individual mitochondrial Ca2+ recording, we discovered impaired extrusion kinetics in mitochondria fragmented by both Drp1 and Bβ2 overexpression. Ca2+ uptake was comparable to that of control. Extreme elongation of mitochondria via dominant negative Drp1-K38A enhanced recovery. Understanding these observations, however, requires knowledge of the mitochondrial Ca2+ buffering mechanism. Mitochondrial uptake candidates include MCU and ccdc109b. Our neuronal characterization of MCU confirms a role in mitochondrial Ca2+ buffering, but not a requirement; other components must be involved. Ccdc109b remains an inconclusive candidate, but may be an important regulator of MCU. Mitochondrial efflux transporters include Letm1 and NCLX. Though Letm1 observations are hindered by control artifact, preliminary evidence supports a role in extrusion. The role of NCLX is complicated by possible tissue specificity. Functional expression experiments utilizing Na+ free Li+ external solution suggests absence of NCLX in hippocampal neurons; DRG neurons were capable of Li+ exchange. The above observations confirm the significance of mitochondrial Ca2+ extrusion in neuronal survival. Understanding the mechanisms and regulation of mitochondrial Ca2+ transport has the potential to provide novel therapeutic targets in pathologies of excitotoxic etiology.

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.

From birth, throughout the entire lifespan, the myocardium is constituted by an almost fixed number of cardiomyocytes (CMs). Post-natal heart growth occurs through CM hypertrophy, and the cell achieves the adult phenot ype through profound structural, functional and metabolic maturation. Once fully developed, the heart continuously adapts its performance and structure in response to the varying requests of the organism, elicited by changes in intrinsic and environmental conditions. While acute stresses operate through reversible modulation of contractility, CMs subjected to prolonged increase in workload undergo complex structural remodelling, mostly occurring through further growth necessary to sustain the chronic elevation of mechanical load. Depending on the nature, intensity and duration of the hypertrophying stimuli, cardiac remodelling may lead to either the so-called "physiologic" (i.e., in the athletic heart), or "pathologic" hypertrophy (i.e., in pressure overload), the latter resulting, with time, in cardiac dysfunction, heart failure and death. Although the cellular and clinical phenotypes of the two conditions are different, the common tenet is that, in the initial phases, they share the same adaptive mechanisms, including increased sarcomeric deposition and enhanced/preserved contractility, both of which require increased ATP supply. Unsurprisingly, the regulation of mitochondrial function is a critical process for ATP production to match energetic demand during cell growth. Mitochondria are the CM powerhouse, and [Ca2+] operates as a primary dynamic regulator of ATP production. In CMs, Ca2+ influx into the mitochondrial matrix occurs during the systolic elevation in cytosolic Ca2+, and is mediated by the recently identified mitochondrial Ca2+ uniporter complex (MCUC). Mitochondrial Ca2+ uptake is fundamental in the acute modulation of contractility during the fight-or-flight response triggered by β-adrenergic receptor (β-AR) activation. Consistently, deletion of MCU in mice impairs exercise capacity, and reduction of functioning MCU channels in sino-atrial node cells or ventricular CMs, blunts the chronotropic or contractile response, respectively, to β-AR stimulation. Whether changes in the expression of MCUC proteins take place during cardiac diseases and, conversely, the effect of modulating mitochondrial Ca2+ uptake on myocardial remodelling is, at present not known. The aims of this thesis were to: Aim 1) identify the molecular mechanisms involved in the endogenous regulation of MCU; Aim 2) determine whether MCU has a role in physiologic and pathologic cardiac remodelling and Aim 3) develop an experimental model of cultured cardiomyocytes suited for the in vitro characterization of mitochondrial Ca2+ dynamics in prolonged observations. Results. 1. Content of mitochondrial calcium uniporter (MCU) in cardiomyocytes is regulated by microRNA-1 in physiologic and pathologic hypertrophy. In our preliminary experiments, we compared the protein levels of MCU in normal and hypertrophied hearts, and observed that changes in mitochondrial MCU protein density were not accompanied by parallel alteration in its transcriptional levels. This prompted us to investigate whether post-translational regulation of MCU might occur in myocardial remodelling. We thus focused on microRNAs (miRs), which are small, non-coding RNA sequences (18-25 nt) capable of finely tuning the expression of a variety of genes by interfering with either the stability or the translation of mRNA. By bioinformatics analysis that identified several microRNAs predicted to target MCU 3’UTR (untranslated region). Among these, we focused on miR-1 for its muscle specific expression, the critical role in the activation in cardiac hypertrophy, its conserved homology among species, and its specificity for MCU among MCUC members. Luciferase assay confirmed the prediction and identified the specific seed sequences on the MCU gene. Consistently, CMs expressing miR-1 showed decreased MCU protein content, with no alterations in mRNA expression, which resulted in significant reduction in mitochondrial Ca2+ uptake. We thus investigated whether MCU content was modulated in hypertrophic conditions associated to changes in miR-1, including: i) post-natal development, ii) moderate exercise and iii) pressure overload. By comparing neonatal and adult mouse hearts we observed that, in line with its role of repressor of fetal gene program, miR-1 expression increased during postnatal development and, coherently, MCU protein content decreased without alterations at transcriptional level. Moreover, this modulation was specific for MCU among the molecular components of MCUC, with the exceptions of mitochondrial calcium uniporter b (MCUb) mRNA, which increased. We then investigated the miR-1/MCU axis, in murine and human heart models of physiologic and pathologic hypertrophy. Physiologic hypertrophy was obtained in mice with chronic exercise protocol, which caused enlargement in cardiac size, CM cross-sectional area, and a slight increase in contractility. As compared to sedentary littermates, miR-1 expression level decreased and, consistently, MCU protein content increased. Analysis of the uniporter complex biochemistry in hearts undergone pressure-overload through transverse aortic constriction (TAC) surgery demonstrated that during the initial, compensatory hypertrophy, characterized by modest CM growth with no contractile failure, changes in miR-1 and MCU were similar to those observed in hearts from exercised mice. Remarkably, the reciprocal miR-1 and MCU modulation occurred in a clinically relevant model of cardiac hypertrophy, as shown by the analysis of human heart biopsies obtained from healthy subjects and patients with aortic stenosis-induced hypertrophy. These results suggest that, regardless of the nature of the hypertrophic stimulus (physiologic or pathologic), the initial CM adaptation to increased heart work is characterized by similar enhancement in the availability of uniporter-forming MCU molecules. Given that similar changes in the miR1/MCU axis were detected both upon exercise and compensated pathologic hypertrophy, we made the hypothesis that a common regulatory mechanism may exist. We thus focused on the β-AR system, the primary physiologic mechanism engaged in response to increased heart load, a condition in common between exercise and TAC-induced pressure-overload. Activation of β-AR signalling leads to enhancement of cytosolic Ca2+ oscillations and mitochondrial Ca2+ uptake, and is involved in the parallel activation of hypertrophic pathways, such as the Akt-FOXO cascade. Interestingly, miR-1 expression has been shown to depend on FOXO3a, suggesting that in conditions of chronic β-AR activation, the blockade of FOXO3a nuclear translocation may inhibit miR-1 increase. In support of this hypothesis, treatment of mice undergone TAC with the β1-blocker metoprolol ablated miR-1 repression and prevented accordingly the increase in MCU protein content. Altogether, our data identifies miR-1 as a novel post-translational regulator of MCU, and supports that the miR-1/MCU axis is involved in physiologic and pathologic myocardial remodelling. Future experiments will be aimed at exploiting the mechanicism of miR-1 action on MCU, as well as understanding the complete signalling pathway involved in MCU modulation. Given that miRs are well-suited therapeutic targets, as they can easily be mimicked or antagonized pharmacologically (miR mimics or antagomiRs, respectively), even with target selectivity, our study of the miR-1/MCU axis may open to the refinement of the current therapeutic approaches to treat myocardial hypertrophy. 2. MCU participates to the myocardial adaptation to hypertrophic stimuli. The observation that MCU protein content drops during long-term TAC, in which maladaptation occurs, suggested that MCU protein content fluctuates during pathologic hypertrophy. This led us to investigate whether MCU may have a role in myocardial remodelling caused by chronic increase in cardiac workload. To test this hypothesis, we sought to characterize functionally, biochemically, and morphologically the effect of modulating MCU expression level prior to exposing hearts to pressure overload through TAC. To increase the insight on cellular signalling, we used adrenergic receptor agonists to study the effect of prolonged adrenergic stimulation in cultured CMs. To study the role of MCU in cardiac adaptation to hypertrophy in vivo, we efficiently overexpressed or downregulated MCU, via AAV9 injection. Altering MCU expression did not affect cardiac structure and performance at baseline. However, in mice undergone TAC, MCU overexpression resulted in enhanced hypertrophy, as demonstrated by higher increase in cardiac mass, as compared to TAC-operated WT TAC (injected with AAV9-Empty vector). Interestingly, hypertrophic remodelling had characteristics similar to that of physiologic hypertrophy (i.e. increased capillary density, reduced fibrotic remodelling, and preserved cardiac contractility) also in the advanced stages of hypertrophy (i.e. 8 weeks). On the contrary, silenced mice subjected to TAC displayed a dramatic phenotype caused by the rapid appearance of severe maladaptive remodelling with typical hallmarks of dilated cardiomyopathy, including reduced capillary density, massive replacement fibrosis and decreased cardiac function. Altogether, these processes result in HF and increased susceptibility to sudden cardiac death already four weeks after TAC. To gain insight on the molecular mechanism whereby changes in MCU impact on stress-induced CM growth, we used neonatal rat CMs in which MCU overexpression or downregulation were obtained with adenoviral vectors. Consistently, MCU overexpression and downregulation resulted in enhanced and reduced mitochondrial calcium uptake, respectively. Interestingly, while MCU overexpression did not affect CM size and morphology at baseline, MCU KD cells displayed a significant increased area and disarranged sarcomeres. To mimic the increased sympathetic tone that characterises both physiologic and pathologic hypertrophy, we treated CMs with the onset of both adrenergic agonist norepinephrine (NE). Interestingly, MCU OE cells had a significantly enhanced increase in cell size growth. Conversely, MCU KD cells had a remarkably divergent phenotype, characterized by sarcomere disarray and activation of apoptosis. These data were intriguingly similar to the phenotype observed in MCU KD hearts developing dilated cardiomyopathy after TAC. The following analyses regarded the activation state of several pro-hypertrophic pathways. Interestingly, MCU overexpression determined faster activation of calcineurin/NFAT pathway upon adrenergic stimulation. Our data point at the participation of Akt/GSK3axis in NFAT enhanced nuclear translocation, presumably downstream of CaMKII-mediated Akt phosphorylation. Indeed, inhibition of CaMKII in MCU OE cardiomyocytes resulted in hypertrophic growth comparable to control cells. To conclude, our studies show that increased heart workload, as achieved in vivo by TAC and mimicked in cells by NE treatment, is well tolerated when MCU levels are augmented by overexpression. Conversely, MCU downregulation leads, in the same conditions, to cell death and consistently faster maladaptive cellular and tissue remodelling. These data are well in accord with our preliminary observation that MCU content, increased in the compensated hypertrophy, decreases in the advanced remodelling associated to HF. Second, we have identified the AR/CaMKII/Akt cascade as a key signalling pathway involved in myocardial hypertrophy and dependent on MCU modulation. 3. In vitro maturation of cultured neonatal cardiomyocytes. Primary neonatal CMs are a widely used cellular model in molecular cardiology, which can be maintained in culture for several days and is easily amenable to genetic manipulation. However, this cell type has important functional and structural differences with the mature CMs. These differences range from the expression of different myosin isoforms to maximize contractile performance, to changes in metabolism allowing increased ATP production to sustain higher consumption. Importantly, postnatal cellular maturation involves structures that regulate Ca2+ dynamics. In particular, in neonatal cells contraction is mostly due to Ca2+ entering through the plasmalemmal L-type Ca2+ channels (LTCC), directly triggering the activation of the sarcomeres, with little contribution from intracellular Ca2+ release from the immature SR stores. In contrast, in adult cells the plasma membrane has fully developed invaginations known as T-tubules which face the terminal SR cisternae, so that LTCC are in close juxtaposition to the Ca2+ Release Units (CRU) formed by the intracellular Ca2+ release channel, ryanodine receptor (RyR). Such arrangement allows few Ca2+ ions entering the cell to trigger release of further Ca2+ from the SR, in a process known as Ca2+-Induced-Ca2+-Release (CICR), which drives contraction. In parallel with the development of SR, the mitochondrial population enriches and interfibrillary mitochondria tether to the SR, in proximity to the CRUs, a condition in which the organelle is found within the confines of a high Ca2+ microdomain, fundamental to drive the ion into the mitochondrial matrix. With these notions in mind, we sought to develop a protocol promoting maturation of neonatal CMs, thus obtaining a cellular model better suited to the study of subcellular Ca2+ handling in order to identify the mechanisms linking mitochondrial Ca2+ dynamics to hypertrophic remodelling. To induce maturation of neonatal CMs, we modified the composition of the media traditionally used to maintain cells in culture. By removing serum from the culture medium, we could avoid cell proliferation and de-differentiation. In addition, we reduced glucose content and added vitamin co-factors and trophic hormones, such as insulin, to compensate the absence of mammalian serum. Furthermore, we improved the preparation purity by eliminating contaminating cardiac fibroblasts, which secrete growth factors and matrix components, promoting cell de-differentiation and hyperplastic growth. With these changes in the isolation conditions, we obtained a pure population of CMs that can be maintained in culture for several weeks, and after few days already acquired a different morphology, compared to those obtained with the more commonly used protocol. Indeed, microscopy imaging showed that the cells were larger, rectangular-shaped, with a regular perimeter, lacking the typical ramifications of neonatal CMs, and a higher long/short axis ratio. Moreover, we observed an increased area occupied by the contractile apparatus, which appeared more regularly displaced. Mitochondria appeared longitudinally displaced along and between the sarcomeres, similarly to adult cells. In addition, immunostaining of RyRs revealed that the protein appeared in clusters more regularly distributed, thus mimicking the phenotype observed in fully differentiated CMs and suggesting increased maturation of the SR. In line with this, we observed shorter and smaller Ca2+ sparks, which are elementary Ca2+ signalling events depending on RyR opening, thus supporting that the more organized RyR clusters formed functionally active CRU, alike those of more mature cells. Interestingly, cells were more receptive to adrenergic agonists, displaying a more pronounced growth by hypertrophy as compared to traditional neonatal CMs. All the aforementioned aspects demonstrate that these cells may represent an in vitro model system well-suited to the study of Ca2+ dynamics and its relation with hypertrophic growth. Remarkably, these properties did not compromise the amenability for genetic manipulation, either via viral infection or transient plasmid transfection. Future experiment will aim at fully characterizing the Ca2+-related structures, such as T-Tubules, as well as formation of dyads.
Dal momento della nascita, per tutta la durata della vita, il miocardio è costituito da un numero pressoché fisso di cardiomiociti (CM). Infatti, la crescita postnatale del cuore è di tipo ipertrofico, per cui lo sviluppo del cardiomiocita, anch’esso di tipo ipertrofico, avviene attraverso un profondo rimodellamento strutturale, funzionale e metabolico. Una volta raggiunto un completo sviluppo, il cuore adatta continuamente la sua contrattilità e struttura in base alle richieste perfusionali dell’organismo, che variano in base a fattori intrinseci ed ambientali. Stimoli acuti determinano la modulazione della contrattilità, mentre stimoli cronici, che richiedono una performance elevata nel tempo, fanno sì che i cardiomiociti rimodellino la loro struttura, crescendo ulteriormente per sostenere l’aumentato carico meccanico. In base a tipo, intensità e durata dello stimolo ipertrofico, il rimodellamento cardiaco può portare ad ipertrofia fisiologica (come nel caso del “cuore d’atleta”) o patologica (ad esempio nel sovraccarico pressorio): in quest’ultimo caso, la crescita ipertrofica risulterà nel tempo in scompenso cardiaco, insufficienza cardiaca e morte. Nonostante i fenotipi cellulare e clinico siano distinti, il comune denominatore di queste condizioni è che, nelle fasi iniziali, i processi sono di tipo adattativo e comprendono la deposizione di nuovi sarcomeri nei cardiomiociti, per garantire una contrattilità migliorata o quanto meno preservata. Queste proprietà richiedono entrambe una maggiore produzione di ATP. Non sorprende quindi il fatto che la regolazione della funzione mitocondriale sia un processo critico per la produzione di ATP, per soddisfare il fabbisogno energetico durante la crescita ipertrofica. I mitocondri sono la “centrale energetica” della cellula e la concentrazione di Ca2+ opera come un regolatore dinamico primario della produzione di ATP. Nei cardiomiociti, l’influsso di Ca2+ nella matrice mitocondriale avviene durante l’aumento di Ca2+ sistolico ed è mediato dal complesso dell’uniporto mitocondriale per il calcio (MCUC), recentemente identificato. L’uptake di Ca2+ mitocondriale è un processo fondamentale nella modulazione acuta della contrattilità durante la risposta “fight-or-flight” attivata dall’attivazione dei recettori ß-adrenergici. A prova di ciò, la delezione di MCU nel modello murino diminuisce la capacità d’esercizio7, mentre la riduzione di MCU nelle cellule del nodo senoatriale o dei ventricoli riduce le risposte cronotropiche o contrattili, rispettivamente, indotte dalla stimolazione ß-adrenergica. Al momento non è noto se avvengano cambi nell’espressione delle proteine formanti MCUC in diverse situazioni fisiopatologiche, così come non è noto l’effetto della modulazione dell’uptake di Ca2+ mitocondriale durante il rimodellamento cardiaco. Su queste basi, gli obiettivi del mio progetto di dottorato sono: 1) Identificare i meccanismi molecolari coinvolti nella regolazione endogena di MCU; 2) Determinare se MCU ha un ruolo nel rimodellamento fisiologico e patologico del cuore; 3) Sviluppare un modello sperimentale di cardiomiociti isolati da cuori neonati per la caratterizzazione in vitro delle dinamiche del Ca2+mitocondriale su tempi prolungati. Risultati. 1. Il contenuto dell’uniporto mitocondriale per il calcio (MCU) nei cardiomiociti è dinamicamente regolato da miR-1 nell’ipertrofia fisiologica e patologica. In esperimenti preliminari condotti nel nostro laboratorio, abbiamo confrontato i livelli proteici di MCU in cuori normali ed ipertrofici, ed abbiamo osservato che le variazioni nel contenuto proteico di MCU non erano accompagnate da variazioni in parallelo del suo trascritto. Ciò ci ha portato ad investigare se, nel rimodellamento cardiaco, potesse avvenire una regolazione post-trascrizionale di MCU. Ci siamo così focalizzati sui microRNA (miR), piccole sequenze non codificanti di RNA (18-25 nucleotidi) capaci di modulare finemente l’espressione di svariati geni, grazie all’interferenza con la stabilità o la traduzione dell’mRNA target. Un numero crescente di evidenze rivela il ruolo fondamentale dei miRs nell’ipertrofia cardiaca e, in altri tessuti, è stato dimostrato come certi miR regolino il contenuto di MCU. Tramite ricerca bioinformatica, abbiamo identificato diversi microRNA che potrebbero appaiarsi alla regione 3’UTR di MCU. Tra questi, ci siamo focalizzati su miR-1 per la sua espressione muscolo-specifica, il suo ruolo critico nell’ipertrofia cardiaca, la sua omologia conservata tra diverse specie e la specificità per MCU tra i membri del complesso MCUC. Il saggio di luciferasi ha confermato quanto predetto dalla bioinformatica ed ha permesso di identificare specifiche sequenze complementari sul gene di MCU. Consistentemente, cardiomiociti over-esprimenti miR-1 hanno mostrato un diminuito contenuto proteico di MCU senza alterazioni nel suo mRNA, risultando in una riduzione significativa nella capacità di importare Ca2+ nella matrice mitocondriale. Quindi, abbiamo testato l’ipotesi che il contenuto di MCU fosse modulato in condizioni di ipertrofia associate a variazioni nell’espressione di miR-1, quali: i) lo sviluppo postnatale, ii) l’esercizio moderato, iii) il sovraccarico pressorio. Confrontando cuori neonati ed adulti abbiamo osservato che l’espressione di miR-1 aumenta, in linea col suo ruolo di repressore del programma genico fetale. Questo calo di miR-1 è accompagnato da un aumento nel contenuto proteico di MCU senza che ne aumentasse il trascritto. Inoltre, abbiamo osservato come solo il contenuto di MCU vari, tra i vari membri del complesso, eccezion fatta per l’mRNA di MCUb, che aumenta. Quindi, abbiamo analizzato l’asse miR-1/MCU in cuori ipertrofici murini e umani, con rimodellamenti sia fisiologici che patologici. Nei topi, l’ipertrofia fisiologica è stata indotta tramite protocollo di esercizio cronico, efficace nel determinare ingrandimento cardiaco, dei singoli cardiomiociti ed un aumento della contrattilità35. Il confronto coi cuori di topi sedentari ho dimostrato come il livello di miR-1 scenda nell’esercizio e, consistentemente, quello proteico di MCU salga. L’analisi del complesso in cuori sottoposti a costrizione aortica ha dimostrato come, durante l’iniziale fase compensata, caratterizzata da crescita dei cardiomiociti senza scompenso, le variazioni di miR-1 e MCU rispecchino quelle osservate nei topi esercitati. Inoltre, le reciproche variazioni di miR-1 e MCU accadono anche in un modello di ipertrofia di rilevanza clinica, come dimostrato dalle analisi di biopsie cardiache umane provenienti da donatori sani e pazienti con ipertrofia causata da stenosi aortica. Questi risultati indicano che, indipendentemente dalla natura dello stimolo ipertrofico (fisiologico o ipertrofico), l’iniziale adattamento cardiaco all’aumentata richiesta contrattile è caratterizzato da analoghi aumenti nella disponibilità cellulare di MCU. Viste le variazioni analoghe dell’asse miR-1/MCU riscontrate sia in ipertrofia indotta da esercizio che in quella compensata patologica, abbiamo ipotizzato che ci sia un meccanismo regolatorio comune. Ci siamo così focalizzati sul sistema ß-adrenergico, il primo meccanismo fisiologico coinvolto nella risposta all’aumentato carico di lavoro, condizione che accomuna sia ipertrofia da esercizio che da costrizione aortica. L’attivazione del signalling ß-adrenergico, infatti, determina aumento delle oscillazioni di Ca2+ citosolico e conseguentemente dell’uptake mitocondriale. In parallelo, l’attivazione di queste cascate di segnale è coinvolta nell’attivazione di vie di segnale di ipertrofia come Akt-FOXO. È interessante notare che l’espressione di miR-1, come è stato dimostrato, dipende da FOXO3a, indicando che, in condizioni di attivazione cronica dei recettori ß-adrenergici, il blocco della traslocazione nucleare di FOXO3a potrebbe inibire l’aumento di miR-1. Per supportare questa ipotesi, abbiamo trattato topi sottoposti a costrizione aortica col ß-bloccante metoprololo che, in linea con quanto ipotizzato, è stato in grado di abolire la repressione di miR-1 e di conseguenza l’aumento di MCU. Conclusioni e prospettive future. Complessivamente, i nostri dati identificano miR-1 come un nuovo regolatore post-trascrizionale di MCU e supportano l’idea che l’asse miR-1/MCU sia coinvolto nel rimodellamento ipertrofico fisiologico e patologico. Esperimenti futuri mireranno ad approfondire il ruolo causale di miR-1 nella modulazione di MCU, ed a identificare la via molecolare coinvolta nel processo. Attualmente esistono tools farmacologici (quali miR-mimics o antagomiRs) in grado di interagire coi miR endogeni, antagonizzandoli o sostituendoli, modulando con efficacia e selettività l’espressione degli mRNA target. Su queste basi, il nostro studio sull’asse miR-1/MCU può aprire a nuove prospettive terapeutiche per trattare l’ipertrofia cardiaca. 2. MCU partecipa all’adattamento cardiaco a stimoli ipertrofici. L’osservazione di come il contenuto di MCU cali durante la fase maladattativa dell’ipertrofia patologica, suggerisce che esso fluttui nelle varie fasi dell’ipertrofia. Questa osservazione ci ha indotto a cercare di determinare se MCU potesse avere un ruolo attivo nel rimodellamento cardiaco. Per testare quest’ipotesi, abbiamo modulato il livello di MCU in topi successivamente sottoposti a sovraccarico pressorio. Inoltre, per avere dettagli meccanicistici sul signalling cellulare, abbiamo modulato l’espressione di MCU in vitro, e abbiamo studiato l’effetto della sua overespressione o silenziamento nella risposta ad incubazione cronica con agonisti adrenergici. Per studiare il ruolo di MCU nell’adattamento cardiaco in vivo, abbiamo overespresso o silenziato l’uniporto mediante l’uso di vettori virali (AAV9). La modulazione di MCU, per sé, non ha alterato la struttura e la performance cardiaca. Tuttavia, quando abbiamo sottoposto gli animali a TAC, abbiamo osservato come l’overespressione di MCU comporti aumentata crescita ipertrofica, confrontando con animali WT allo stesso tempo dopo l’inizio della costrizione aortica. Inoltre, il rimodellamento nei topi overesprimenti ha caratteristiche simili a quello dell’ipertrofia fisiologica, quali aumentata densità capillare, scarsa fibrosi, funzionalità cardiaca preservata anche dopo 8 settimane di sovraccarico pressorio. Al contrario, il silenziamento di MCU ostacola l’adattamento cardiaco all’aumentata pressione, determinando un maladattamento prematuro, con caratteristiche tipiche della cardiomiopatia dilatativa, quali ridotta densità capillare, fibrosi diffusa ed inadeguata contrattilità. Queste caratteristiche hanno portato i topi MCU silenziati a sviluppare scompenso ed insufficienza cardiaca, ed a morire dopo solo 4 settimane dalla TAC. Per approfondire i meccanismi molecolari mediante i quali MCU impatta nella crescita ipertrofica dei cardiomiociti, abbiamo overespresso o silenziato MCU in cardiomiociti neonatali di ratto. Eseguendo esperimenti di live imaging delle dinamiche di Ca2+ mitocondriali con la sonda “mito-CaMeleon”, abbiamo appurato come la modulazione di MCU risulti in aumentato o diminuito uptake di Ca2+ mitocondriale. Se da un lato l’over-espressione di MCU non determina alterazioni morfologiche in condizioni basali, cellule silenziate dimostrano dimensioni maggiori rispetto a cellule di controllo, con evidente alterazioni nella struttura sarcomerica. Per mimare l’iperattivazione del sistema nervoso simpatico che si riscontra nell’ipertrofia sia fisiologica che patologica, abbiamo incubato le cellule con norepinefrina. Anche in questo caso, l’overespressione di MCU aumenta la crescita ipertrofica, mentre il suo silenziamento ha un effetto opposto, contraddistinto da compromissione dei sarcomeri ad attivazione di apoptosi, in evidente analogia ai dati ottenuti in vivo. Le successive analisi sono state mirate per approfondire lo stato di attivazione di divere vie di segnale medianti ipertrofia. Abbiamo rilevato come l’overespressione di MCU, in cardiomiociti sottoposti a stimolazione adrenergica, acceleri l’attivazione dell’asse calcineurina/NFAT. Inoltre, i nostri dati suggeriscono la partecipazione dell’asse Akt/ GSK3ß all’aumentata attivazione di NFAT, in una cascata presumibilmente a valle di CaMKII che fosforila Akt. Infatti, l’inibizione di CaMKII in cardiomiociti MCU overesprimenti determina una crescita ipertrofica comparabile a cellule di controllo. Per concludere, i nostri risultati dimostrano come l’aumento del carico cardiaco, indotto in vivo da TAC ed in vitro da trattamento con noradrenalina, sia ben tollerato quando i livelli di MCU sono aumentati dall’overespressione. Al contrario, il silenziamento di MCU induce, nelle stesse condizioni, morte cellulare e prematuro rimodellamento maladattativo. Questi dati sono in accordo con le nostre osservazioni preliminari che indicano come il contenuto proteico di MCU, che aumenta nell’ipertrofia compensata, diminuisca nel successivo rimodellamento patologico che determina scompenso cardiaco. Inoltre, abbiamo identificato l’asse ß-AR/CaMKII/Akt come cruciale nell’ipertrofia cardiaca e dipendente dalla modulazione di MCU. 3. Sviluppo di un protocollo di coltura che induca la maturazione di cardiomiociti neonatali in vitro Le colture primarie di cardiomiociti neonatali sono un modello cellulare ampiamente utilizzato nella cardiologia molecolare, in quanto possono esser mantenuti in coltura per più giorni e sono facilmente manipolabili geneticamente28. Tuttavia, questo tipo cellulare ha importanti differenze funzionali e strutturali rispetto ai cardiomiociti adulti. Queste differenze vanno dall’espressione di diverse isoforme di miosina (nel topo, dalla ß alla α), necessario per ottimizzare la performance contrattile, a cambi nel metabolismo (che passa da glucidico ad ossidativo), in modo da garantire maggior apporto di ATP in vista di un maggior consumo29. Inoltre, il processo di maturazione postnatale delle cellule comprende alterazioni nelle strutture coinvolte nelle dinamiche di Ca2+ 30. In particolare, nelle cellule neonatali, la contrazione avviene principalmente grazie al Ca2+ che entra dai canali del Ca2+ di tipo L situati nella membrana citoplasmatica. Il Ca2+ che entra attiva direttamente i sarcomeri, con un minimo contributo del Ca2+ contenuto nelle vescicole che costituiscono un immaturo reticolo sarcoplasmatico. Al contrario, nelle cellule adulte la membrana plasmatica ha sviluppato una serie di invaginazioni note come tubuli T che penetrano nella cellula e giungono all’estremità del reticolo sarcoplasmatico, ora costituito dal tipico sistema di cisterne, cosicché i canali del Ca2+ di tipo L siano a stretto contatto coi RyR2, formando cosi le Unità deputate al Rilascio del Ca2+ (CRUs). Questa sofisticata struttura fa sì che le poche molecole di Ca2+ che entrano dai canali nei tubuli T possano scatenare il Rilascio di Ca2+ indotto dal Ca2+ (CICR), determinando l’uscita di un’ingente quantità di ione dal reticolo sarcoplasmatico. Un altro importante cambiamento interessa i mitocondri che, se nel cardiomiocita neonatale occupano principalmente la zona perinucleare, in quello adulto si dispongono anche negli spazi sub-sarcolemmali ed inter-miofibrillari. In questi distretti, i mitocondri sono in prossimità del reticolo sarcoplasmatico, al quale possono ancorarsi fisicamente, trovandosi così in distretti cellulari caratterizzati da elevate concentrazioni di Ca2+. Tenendo a mente questi fattori, il nostro obiettivo è stato quello di sviluppare un protocollo che promuovesse la maturazione di cardiomiociti neonatali verso un fenotipo adulto, ottenendo così un modello sperimentale ottimale per lo studio delle dinamiche del Ca2+ cellulare, ed identificare così i meccanismi che connettono il Ca2+ mitocondriale al rimodellamento ipertrofico. Per indurre la maturazione dei cardiomiociti neonatali abbiamo modificato la composizione dei terreni di coltura tradizionalmente usati. Per mantenere le cellule ad una concentrazione di glucosio simile a quella fisiologica, abbiamo cambiato il costituente principale del terreno, passando da DMEM (Dulbecco’s modified eagle medium) a MEM (minimum essential medium) e riducendo così la concentrazione da 25 mM a 5 mM, valore, quest’ultimo, paragonabile alla concentrazione fisiologica in vivo. Per ridurre la proliferazione dei fibroblasti, che tramite secrezione di fattori di crescita e componenti della matrice extracellulare determinerebbero de-differenziamento dei cardiomiociti, abbiamo fortemente ridotto il quantitativo di siero ed aggiunto un agente proliferativo (BrdU). Per compensare la rimozione del siero, abbiamo aggiunto co-fattori vitaminici ed ormoni trofici, come l’insulina. In tal modo abbiamo ottenuto una popolazione pura di cardiomiociti che può essere tenuta in coltura per più settimane, e che già dopo pochi giorni mostrano una morfologia diversa dalle cellule ottenute col protocollo tradizionale. Analisi alla microscopia hanno evidenziato come queste cellule siano più grandi, rettangolari con un asse maggiore ben distinto da un asse minore, ed un perimetro regolare senza le tipiche ramificazioni dei cardiomiociti immaturi neonatali. A livello subcellulare, abbiamo osservato una maggiore estensione dell’apparato contrattile, rivelatosi disposto in maniera più regolare. I mitocondri appaiono disposti longitudinalmente accanto e tra i sarcomeri, come nelle cellule adulte. Inoltre, l’immunofluorescenza per il recettore rianodinico ne ha evidenziato la presenza in clusters, distribuiti in maniera regolare, in analogia alla loro distribuzione in cellule mature, suggerendo così la presenza di un reticolo sarcoplasmatico maggiorente formato. Consistentemente con ciò, abbiamo osservato minori e più rapidi Ca2+ sparks, eventi elementari di dinamiche di calcio, determinati dall’apertura transiente di RyR. La minore frequenza ed entità di questi sparks suggerisce che i RyR disposti in maniera regolare in clusters determini la formazione di vere e proprie unità deputate al rilascio di calcio (Calcium Release Units, CRUs), strutture fondamentali nei cardiomiociti adulti. Infine, queste cellule han risposto maggiormente al trattamento con agonisti adrenergici, riportando una crescita ipertrofica maggiore rispetto a cellule neonatali tradizionali sottoposte allo stesso trattamento. Tutte queste caratteristiche sopracitate indicano come queste cellule possano rappresentare un modello in vitro adatto allo studio delle dinamiche di Ca2+ intracellulare, specialmente nel rimodellamento ipertrofico. È importante sottolineare come questo maggior grado di maturazione dei cardiomiociti neonatali non sia a discapito della capacità di manipolarli geneticamente, con tecniche di trasfezione od infezione. Esperimenti futuri cercheranno di caratterizzare a fondo le strutture coinvolte nelle dinamiche di calcio intracellulari, come ad esempio la formazione di Tubuli T ed il rapporto di questi con il reticolo sarcoplasmatico ed i mitocondri.

Altered cardiac energetics and calcium handling are characteristic features of cardiovascular disease. Mitochondria play a significant role in both cellular energy generation and calcium homeostasis and may be a key integration point of these two systems. Calcium uptake into mitochondria occurs via a recently identified mitochondrial calcium uniporter complex. In the first part of this thesis, I characterize the phylogenomic distribution of the uniporter’s membrane spanning pore (MCU) and regulatory subunits (MICU1 and MICU2). Homologs of both MCU and MICU1 tend to co-occur in all major branches of eukaryotic life but both have been lost along certain protozoan and fungal lineages. MICU2 represents a recent duplication of MICU1. Several bacterial genomes also contain putative MCU homologs that may represent prokaryotic calcium channels. The analyses indicate that the uniporter may have been an early feature of mitochondria. In the second part of this thesis, I perform transcriptome wide analysis of human and mouse cardiomyopathy datasets and identify MICU2, a regulatory component of the mitochondrial calcium uniporter, as one of six genes consistently upregulated in cardiac disease states. I test the hypothesis that increased MICU2 expression is cardio-protective by generating a global Micu2-/- mouse. These mice have diastolic dysfunction. Isolated Micu2-/- cardiomyocytes show altered sarcomere relaxation and cytosolic calcium reuptake kinetics and Micu2-/- ventricular tissue has transcriptional dysregulation of genes encoding sarcomere proteins and bZIP transcription factors. When exposed to two weeks of angiotensin 2, a pharmacologic hypertrophic stimuli, Micu2-/- mice exhibit both systolic and diastolic dysfunction. Together, these data point to a significant and previously unappreciated role for Micu2 in maintaining both cardiac and vascular homeostasis.

A long held hypothesis in mitochondrial biology holds that increases in mitochondrial Ca2+ levels stimulate the activity of matrix dehydrogenases that catalyze production of NADH and eventually donate electrons to electron transport in order to increase ATP formation. At the same time, mitochondrial Ca2+ overload is a deleterious event leading to opening of the mitochondrial permeability transition pore, increasing reactive oxygen species and initiating pathways that contribute to cell death. These fundamental hypotheses are best studied in the heart because of the critical energy supply-demand relationship in myocardium, but were untestable in vivo until the discovery of the mitochondrial Ca2+ uniporter (MCU). The molecular identity of the MCU pore forming subunit was recently discovered, which allowed me to study a transgenic mouse with myocardial delimited expression of a dominant negative MCU. My lab developed mice with myocardial-delimited transgenic expression of a dominant negative MCU to test these fundamental hypotheses and to determine how MCU controls physiological and pathological stress responses in vivo, ex vivo, and in situ. My studies provide new, unanticipated information that contributes to our understanding the relationship between mitochondrial Ca2+, oxygen utilization, cardiac pacemaking and pathologic stress responses in heart. Here, I show that mice with myocardial-targeted MCU inhibition have hearts with surprisingly high oxygen consumption rates due to elevated cytoplasmic Ca2+ in response to physiological stress. Loss of MCU effectively preserved inner mitochondrial membrane potential and prevented an oxidative burst thought to drive myocardial injury and death, but nevertheless failed to protect myocardium from ischemia-reperfusion injury. Increases in oxygen consumption, elevation in cytoplasmic Ca2+ and transcriptional reprogramming mitigate the protective actions of MCU inhibition in vivo. Mice with myocardial selective MCU inhibition have a reduced response to isoproterenol-induced heart rate increase but have normal baseline heart rates. My studies provide novel insight into how MCU contributes to myocardial Ca2+ homeostasis, metabolism, and transcription leading to surprising actions on physiological and pathophysiological responses in heart.

Orientadores: Tiago Rezende Figueira, Roger Frigério Castilho
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
Made available in DSpace on 2018-08-27T03:45:16Z (GMT). No. of bitstreams: 1 Chweih_Hanan_M.pdf: 1684363 bytes, checksum: edae156378f90e7315bca30c16544071 (MD5) Previous issue date: 2015
Resumo: Algumas das características das mitocôndrias, incluindo as suas funções de transporte de Ca2+, podem apresentar dimorfismo sexual e especificidades teciduais. No entanto, as mensurações do transporte de Ca2+ em mitocôndrias isoladas estão sujeitas a artefatos secundários a abertura do poro de transição de permeabilidade mitocondrial (PTP) induzido pelo acúmulo excessivo de Ca2+ nesta organela. Neste estudo, o objetivo inicial foi avaliar se a inibição do PTP pela ciclosporina A (CsA) afeta a mensuração de diversas variáveis que descrevem o transporte de Ca2+por mitocôndrias isoladas de fígado de rato. Os resultados obtidos indicam que as concentrações de estado estável do Ca2+ externo a mitocôndria e as taxas deefluxo mitocondrial de Ca2+através de trocadores seletivos foram superestimados em até 4 vezes quando o PTP não foi inibido farmacologicamente pela CsA. O objetivo subsequente foi analisar o transporte de Ca2+ em mitocôndrias isoladas de fígado, de músculo esquelético, de coração e de cérebro de ratos machos e fêmeas sob condições experimentais específicas (i.e. meio de incubação contendo inibidores TPM, substratos energéticos ligados a NAD e níveis relevantes de Ca2+, Mg2+e Na+). Os dados indicaram que a taxa de influxo de Ca2+em mitocôndrias de fígado foi ~4 vezes superior a dos outros tecidos, as quais foram semelhantes entre si. Em contrapartida, as taxas de efluxo de Ca2+ apresentaram uma maior diversidade entre tecidos, especialmente na presença de Na+. Curiosamente, o efluxo de Ca2+na ausência de Na+foi significativamente mais elevado nas mitocôndrias cardíacas (~4nmol/mg/min) em relação às taxas observadas nos outros tecidos, contrariando a concepção de que o efluxo de Ca2+de mitocôndrias de coração é dependente, quase que exclusivamente, de um trocador que requer Na+. A especificidade em relação ao sexo só foi observada em dois índices relacionados a homeostase mitocondrial de Ca2+(i.e. cinética geral normalizada da captação de Ca2+ e a concentração de estado estável do Ca2+ externo a mitocôndria) em mitocôndrias isoladas de coração (mais lentos ou maiores na fêmea) e na respiração estimulada por ADP em mitocôndrias de fígado (~20% maior na fêmea). O presente estudo demonstrou a importância metodológica de se prevenir a abertura do PTP para a análise das propriedades e da variabilidade fisiológica do transporte de Ca2+por mitocôndrias isoladas. Adicionalmente, concluímos que sob as condições experimentais aqui utilizadas, o efluxo de Ca2+ mitocondrial apresenta grandes especificidades teciduais e que alguns achados desafiam conceitos estabelecidos em estudos anteriores sob condições arguivelmente menos controladas
Abstract: The characteristics of mitochondria, including their Ca2+ transport functions, may exhibit tissue specificity and sex dimorphism. Because the measurements of the Ca2+ handling by isolated mitochondria may be biased by dysfunction secondary to Ca2+-induced mitochondrial permeability transition (MPT) pore opening, this study evaluates the extent to which MPT inhibition by cyclosporine-A affects the measurement of Ca2+ transport in isolated rat liver mitochondria. The results indicate that the steady-state levels of external Ca2+ and the rates of mitochondrial Ca2+ efflux through the selective pathways can be overestimated by up to 4-fold if MPT pore opening is not prevented. Then, we analyzed the Ca2+ transport in isolated mitochondria from the liver, skeletal muscle, heart and brain of male and female rats under incubation conditions containing MPT inhibitors, NAD-linked substrates and relevant levels of free Ca2+, Mg2+ and Na+. Except for the liver mitochondria displaying values4-fold higher, the Ca2+ influx rates were similar among the other tissues. In contrast, the Ca2+ efflux rates exhibited more tissue diversity, especially in the presence of Na+. Interestingly, the Na+-independent Ca2+ efflux was highest in the heart mitochondria (~4 nmol/mg/min), thus challenging the view that heart mitochondrial Ca2+ efflux relies almost exclusively on a Na+-dependent pathway. Sex specificity was only observed in two kinetic indexes (i.e. the normalized overall kinetics of Ca2+ uptake and the steady-state levels of external Ca2+) of heart mitochondrial Ca2+ homeostasis (slower or higher in female)and in the ADP-stimulated respiration of liver mitochondria (~20% higher in females). The present study shows the methodological importance of preventing MPT when measuring the properties and the physiological variability of the Ca2+ handling by isolated mitochondria. Moreover, we conclude that mitochondrial Ca2+ efflux exhibits great tissue specificity under our conditions, which may challenge some concepts raised in previous studies that employed experimental conditions that are arguably not well controlled
Mestrado
Fisiopatologia Médica
Mestra em Ciências

Chloroplasts and mitochondria play essential roles in the plant physiology and are emerging as important players in intracellular Ca2+ signaling. A major role in the context of organellar ion homeostasis is played by ion channels. In fact, they are responsible for the regulation of ion distribution between compartments and they are proposed to guarantee Ca2+ signaling, proper osmotic potential, optimal pH for enzymatic activities and electron transport chains’ function. In plants, only a few of channel families have been found to localize to chloroplast/mitochondria and most of them have not been fully characterized. In addition, the molecular mechanisms by which chloroplasts and mitochondria accumulate and release Ca2+ are still far from being clarified. The present work aimed to characterize possible players in organellar Ca2+ flux-mediating systems in plants, namely the Ionotropic Glutamate Receptors (GLRs, non-selective cation channels) and Mitochondrial Calcium Uniporters (MCUs), combining reverse genetics, biochemistry and in vivo localization studies. The research here presented allowed to deepen the study on land plant GLRs, to characterize two novel organellar channels of A. thaliana, i. e. AtGLR3.5 and AtMCU, and to lay the ground for the characterization of the Physcomitrella patens GLRS, PpGLRs, from different points of view. Results revealed additional variability of GLRs among photosynthetic organisms and support the role of GLRs in the regulation of various organellar processes such as photoprotective mechanisms, senescence and mitochondrial structure integrity maintenance. Importantly, the characterization of AtGLR3.5 provided the molecular identification of the first cation channel in plant mitochondria and the characterization of AtMCU1 and MICU allowed for the first time the proposal of a model for the existence and regulation of Ca2+ fluxes in plant mitochondria via MCU complex. The present work therefore contributed to add new knowledge to the field of the regulation of ion homeostasis, especially Ca2+ homeostasis, in mitochondria and chloroplasts.
Cloroplasti e mitocondri hanno un ruolo fondamentale nella fisiologia vegetale e stanno emergendo come importanti attori nel signaling del Ca2+ intracellulare. Un ruolo fondamentale nel contesto dell’omeostasi ionica organellare è giocato dai canali ionici. Infatti essi sono responsabili della regolazione della distribuzione ionica fra compartimenti ed è stato proposto che contribuiscano a garantire il signaling del Ca2+, l’appropriato potenziale osmotico, il pH ottimale per le attività enzimatiche e il funzionamento delle catene di trasporto elettronico. Nelle piante, solo poche famiglie di canali sono state identificate in cloroplasti/mitocondri e la maggior parte di loro non è stata pienamente caratterizzata. Inoltre, il meccanismo molecolare attraverso cui cloroplasti e mitocondri accumulano e rilasciano Ca2+ è ancora lontano dall’essere chiarito. Lo scopo del presente lavoro è stato quello di caratterizzare possibili attori coinvolti nel nella mediazione dei flussi di Ca2+ organellari, in particolare i recettori ionotropici del glutammato (GLRs) e gli uniporti mitocondriali del calcio (MCU), combinando tecniche di genetica inversa, biochimica e studi di localizzazione in vivo. La ricerca qui presentata ha permesso di approfondire lo studio sui GLR vegetali, di caratterizzare due nuovi canali organellari di A. thaliana, AtGLR3.5 e AtMCU, e di gettare le fondamenta per la caratterizzazione dei GLR di Physcomitrella patens, PpGLR, da diversi punti di vista. I risultati del lavoro hanno rivelato ulteriore variabilità dei GLR fra gli organismi fotosintetici e hanno mostrato come i GLR siano coinvolti nella regolazione di diversi processi organellari come i meccanismi di fotoprotezione, la senescenza e il mantenimento dell’integrità strutturale mitocondriale. La caratterizzazione di AtGLR3.5 ha permesso l’identificazione molecolare del primo canale cationico mitocondriale vegetale e la caratterizzazione di AtMCU1 ha permesso per la prima volta di proporre un modello che preveda l’esistenza e la regolazione dei flussi di Ca2+ nei mitocondri vegetali per mezzo del complesso MCU. Il lavoro qui presentato ha pertanto contribuito ad aggiungere nuova conoscenza al campo della regolazione dell’omeostasi ionica, specialmente l’omeostasi del Ca2+, nei mitocondri e nei cloroplasti.

Following neuronal injury, calcium signaling plays a critical role in promoting repair processes. Injury produces an initial cytosolic calcium elevation mediated by calcium entry from the cut site, plasma membrane channels, and intracellular storage compartments. Subsequently, a variety of signaling factors are involved in promoting growth cone formation and axon outgrowth and guidance, some of which include DLK-1, CaMP, CED-3, CED-4, and calreticulin. Specific proteins mediating calcium transport have also been reported to significantly affect regenerative outgrowth, particularly inositol triphosphate receptors, voltage-gated calcium channels, and ryanodine receptors. Given that mitochondria can store intracellular calcium and regulate cytosolic calcium levels, we hypothesized that the mitochondrial uniporter (MCU) may play a significant role in neuronal regeneration. We found that inhibiting calcium entry into the mitochondria via a loss of function mutation in MCU significantly enhances axonal outgrowth following laser axotomy of single neurons in C. elegans. This effect is calcium-dependent, with the MCU mutant regenerative phenotype reverting to baseline levels when mutants are chronically treated with the calcium chelator EGTA. We also find that sub-cellular calcium signals at the axon cut site are significantly reduced in MCU mutants, while basal levels of calcium and axon guidance remain unaffected. These findings suggest that mitochondrial calcium regulation plays a significant role in the regeneration of single neurons, and that inhibition of MCU activity may be a promising avenue for the treatment of clinical syndromes derived from axonal injury, such as spinal cord injury.
2017-11-03T00:00:00Z