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Campo, Antonio (2018) Role and regulation of the mitochondrial calcium uniporter (MCU) in cardiac adaptation to stresses. [Ph.D. thesis]

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Abstract (english)

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.

Abstract (italian)

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.

EPrint type:Ph.D. thesis
Tutor:Mongillo, Marco
Supervisor:Zaglia, Tania
Ph.D. course:Ciclo 30 > Corsi 30 > SCIENZE BIOMEDICHE SPERIMENTALI
Data di deposito della tesi:12 January 2018
Anno di Pubblicazione:12 January 2018
Key Words:MCU mitochondrial calcium uniporter heart cardiomyocyte hypertrophy
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/46 Scienze tecniche di medicina di laboratorio
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Biomediche
Codice ID:10634
Depositato il:26 Oct 2018 09:13
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