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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.
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.
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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1 Sedmera, D. & Thompson, R. P. Myocyte proliferation in the developing heart. Developmental Dynamics 240, 1322-1334, doi:10.1002/dvdy.22650 (2011). Cerca con Google

2 Badeer, H. S. BIOLOGICAL SIGNIFICANCE OF CARDIAC HYPERTROPHY. The American journal of cardiology 14, 133-138 (1964). Cerca con Google

3 Shimizu, I. & Minamino, T. Physiological and pathological cardiac hypertrophy. Journal of Molecular and Cellular Cardiology 97, 245-262, doi:10.1016/j.yjmcc.2016.06.001 (2016). Cerca con Google

4 Williams, G. S. B., Boyman, L. & Lederer, W. J. Mitochondrial calcium and the regulation of metabolism in the heart. Journal of Molecular and Cellular Cardiology 78, 35-45, doi:10.1016/j.yjmcc.2014.10.019 (2015). Cerca con Google

5 De Stefani, D., Raffaello, A., Teardo, E., Szabò, I. & Rizzuto, R. A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature 476, 336-340, doi:10.1038/nature10230 (2011). Cerca con Google

6 Baughman, J. M. et al. Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature 476, 341-345, doi:10.1038/nature10234 (2011). Cerca con Google

7 Pan, X. et al. The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter. Nature Cell Biology 15, 1464-1472, doi:10.1038/ncb2868 (2013). Cerca con Google

8 Rasmussen, T. P. et al. Inhibition of MCU forces extramitochondrial adaptations governing physiological and pathological stress responses in heart. Proceedings of the National Academy of Sciences 112, 9129-9134, doi:10.1073/pnas.1504705112 (2015). Cerca con Google

9 Kwong, Jennifer Q. et al. The Mitochondrial Calcium Uniporter Selectively Matches Metabolic Output to Acute Contractile Stress in the Heart. Cell Reports 12, 15-22, doi:10.1016/j.celrep.2015.06.002 (2015). Cerca con Google

10 Luongo, Timothy S. et al. The Mitochondrial Calcium Uniporter Matches Energetic Supply with Cardiac Workload during Stress and Modulates Permeability Transition. Cell Reports 12, 23-34, doi:10.1016/j.celrep.2015.06.017 (2015). Cerca con Google

11 Wu, Y. et al. The mitochondrial uniporter controls fight or flight heart rate increases. Nature Communications 6, 6081, doi:10.1038/ncomms7081 (2015). Cerca con Google

12 Small, E. M. & Olson, E. N. Pervasive roles of microRNAs in cardiovascular biology. Nature 469, 336-342, doi:10.1038/nature09783 (2011). Cerca con Google

13 Sayed, D., Hong, C., Chen, I. Y., Lypowy, J. & Abdellatif, M. MicroRNAs Play an Essential Role in the Development of Cardiac Hypertrophy. Circulation Research 100, 416-424, doi:10.1161/01.res.0000257913.42552.23 (2007). Cerca con Google

14 Ikeda, S. et al. MicroRNA-1 Negatively Regulates Expression of the Hypertrophy-Associated Calmodulin and Mef2a Genes. Molecular and Cellular Biology 29, 2193-2204, doi:10.1128/mcb.01222-08 (2009). Cerca con Google

15 Elia, L. et al. Reciprocal Regulation of MicroRNA-1 and Insulin-Like Growth Factor-1 Signal Transduction Cascade in Cardiac and Skeletal Muscle in Physiological and Pathological Conditions. Circulation 120, 2377-2385, doi:10.1161/circulationaha.109.879429 (2009). Cerca con Google

16 Castaldi, A. et al. MicroRNA-133 modulates the beta1-adrenergic receptor transduction cascade. Circ Res 115, 273-283, doi:10.1161/circresaha.115.303252 (2014). Cerca con Google

17 Care, A. et al. MicroRNA-133 controls cardiac hypertrophy. Nat Med 13, 613-618, doi:10.1038/nm1582 (2007). Cerca con Google

18 Wei, Y. et al. Multifaceted roles of miR-1s in repressing the fetal gene program in the heart. Cell Res 24, 278-292, doi:10.1038/cr.2014.12 (2014). Cerca con Google

19 Porrello, E. R. et al. MiR-15 family regulates postnatal mitotic arrest of cardiomyocytes. Circ Res 109, 670-679, doi:10.1161/circresaha.111.248880 (2011). Cerca con Google

20 Stolen, T. O. et al. Interval training normalizes cardiomyocyte function, diastolic Ca2+ control, and SR Ca2+ release synchronicity in a mouse model of diabetic cardiomyopathy. Circ Res 105, 527-536, doi:10.1161/circresaha.109.199810 (2009). Cerca con Google

21 Lu, X. et al. Measuring local gradients of intramitochondrial [Ca(2+)] in cardiac myocytes during sarcoplasmic reticulum Ca(2+) release. Circ Res 112, 424-431, doi:10.1161/circresaha.111.300501 (2013). Cerca con Google

22 Drago, I., De Stefani, D., Rizzuto, R. & Pozzan, T. Mitochondrial Ca2+ uptake contributes to buffering cytoplasmic Ca2+ peaks in cardiomyocytes. Proceedings of the National Academy of Sciences of the United States of America 109, 12986-12991, doi:10.1073/pnas.1210718109 (2012). Cerca con Google

23 Robert, V. et al. Beat-to-beat oscillations of mitochondrial [Ca2+] in cardiac cells. Embo j 20, 4998-5007, doi:10.1093/emboj/20.17.4998 (2001). Cerca con Google

24 Zaglia, T. et al. Cardiac sympathetic neurons provide trophic signal to the heart via beta2-adrenoceptor-dependent regulation of proteolysis. Cardiovasc Res 97, 240-250, doi:10.1093/cvr/cvs320 (2013). Cerca con Google

25 Kumarswamy, R. et al. SERCA2a gene therapy restores microRNA-1 expression in heart failure via an Akt/FoxO3A-dependent pathway. Eur Heart J 33, 1067-1075, doi:10.1093/eurheartj/ehs043 (2012). Cerca con Google

26 Sugden, P. H., Fuller, S. J., Weiss, S. C. & Clerk, A. Glycogen synthase kinase 3 (GSK3) in the heart: a point of integration in hypertrophic signalling and a therapeutic target? A critical analysis. British Journal of Pharmacology 153, S137-S153, doi:10.1038/sj.bjp.0707659 (2009). Cerca con Google

27 Anderson, M. E., Brown, J. H. & Bers, D. M. CaMKII in myocardial hypertrophy and heart failure. Journal of Molecular and Cellular Cardiology 51, 468-473, doi:10.1016/j.yjmcc.2011.01.012 (2011). Cerca con Google

28 Mongillo, M. & Marks, A. R. Models of heart failure progression: Ca2+ dysregulation. Drug Discovery Today: Disease Models 4, 191-196, doi:10.1016/j.ddmod.2007.06.005 (2007). Cerca con Google

29 Anmann, T. et al. Formation of highly organized intracellular structure and energy metabolism in cardiac muscle cells during postnatal development of rat heart. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1837, 1350-1361, doi:10.1016/j.bbabio.2014.03.015 (2014). Cerca con Google

30 Louch, W. E., Koivumäki, J. T. & Tavi, P. Calcium signalling in developing cardiomyocytes: implications for model systems and disease. The Journal of Physiology 593, 1047-1063, doi:10.1113/jphysiol.2014.274712 (2015). Cerca con Google

31 Nielsen, M. S. Myocyte-fibroblast interactions--risky connections. Heart Rhythm 6, 1650-1651, doi:10.1016/j.hrthm.2009.08.025 (2009). Cerca con Google

32 Santiago, J. J. et al. Cardiac fibroblast to myofibroblast differentiation in vivo and in vitro: expression of focal adhesion components in neonatal and adult rat ventricular myofibroblasts. Developmental dynamics : an official publication of the American Association of Anatomists 239, 1573-1584, doi:10.1002/dvdy.22280 (2010). Cerca con Google

33 Xie, Y. et al. Effects of fibroblast-myocyte coupling on cardiac conduction and vulnerability to reentry: A computational study. Heart rhythm 6, 1641-1649, doi:10.1016/j.hrthm.2009.08.003 (2009). Cerca con Google

34 Zhang, X. et al. Cardiomyocyte differentiation induced in cardiac progenitor cells by cardiac fibroblast-conditioned medium. Experimental biology and medicine (Maywood, N.J.) 239, 628-637, doi:10.1177/1535370214525323 (2014). Cerca con Google

35 Stolen, T. O. et al. Interval Training Normalizes Cardiomyocyte Function, Diastolic Ca2+ Control, and SR Ca2+ Release Synchronicity in a Mouse Model of Diabetic Cardiomyopathy. Circulation Research 105, 527-U547, doi:10.1161/Circresaha.109.199810 (2009). Cerca con Google

36 Boyden, P. A., Hirose, M. & Dun, W. Cardiac Purkinje cells. Heart Rhythm 7, 127-135, doi:10.1016/j.hrthm.2009.09.017 (2010). Cerca con Google

37 Bailey, J. C., Lathrop, D. A. & Pippenger, D. L. Differences between proximal left and right bundle branch block action potential durations and refractoriness in the dog heart. Circ Res 40, 464-468 (1977). Cerca con Google

38 Goldberger, A. L., Rigney, D. R. & West, B. J. Chaos and fractals in human physiology. Scientific American 262, 42-49 (1990). Cerca con Google

39 Dobrzynski, H. et al. Structure, function and clinical relevance of the cardiac conduction system, including the atrioventricular ring and outflow tract tissues. Pharmacol Ther 139, 260-288, doi:10.1016/j.pharmthera.2013.04.010 (2013). Cerca con Google

40 Page, E. Quantitative ultrastructural analysis in cardiac membrane physiology. Am J Physiol 235, C147-158 (1978). Cerca con Google

41 Williams, G. S., Boyman, L., Chikando, A. C., Khairallah, R. J. & Lederer, W. J. Mitochondrial calcium uptake. Proceedings of the National Academy of Sciences of the United States of America 110, 10479-10486, doi:10.1073/pnas.1300410110 (2013). Cerca con Google

42 Morita, H., Seidman, J. & Seidman, C. E. Genetic causes of human heart failure. J Clin Invest 115, 518-526, doi:10.1172/jci24351 (2005). Cerca con Google

43 Bers, D. M. Cardiac sarcoplasmic reticulum calcium leak: basis and roles in cardiac dysfunction. Annu Rev Physiol 76, 107-127, doi:10.1146/annurev-physiol-020911-153308 (2014). Cerca con Google

44 Bers, D. M. Cardiac excitation-contraction coupling. Nature 415, 198-205, doi:10.1038/415198a (2002). Cerca con Google

45 Fabiato, A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol 245, C1-14 (1983). Cerca con Google

46 Cheng, H., Lederer, W. J. & Cannell, M. B. Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science 262, 740-744 (1993). Cerca con Google

47 Lehnart, S. E. et al. Stabilization of cardiac ryanodine receptor prevents intracellular calcium leak and arrhythmias. Proceedings of the National Academy of Sciences of the United States of America 103, 7906-7910, doi:10.1073/pnas.0602133103 (2006). Cerca con Google

48 Wehrens, X. H. et al. FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death. Cell 113, 829-840 (2003). Cerca con Google

49 Lehnart, S. E. et al. Sudden death in familial polymorphic ventricular tachycardia associated with calcium release channel (ryanodine receptor) leak. Circulation 109, 3208-3214, doi:10.1161/01.cir.0000132472.98675.ec (2004). Cerca con Google

50 Huxley, H. E. The mechanism of muscular contraction. Science 164, 1356-1365 (1969). Cerca con Google

51 Egger, M. & Niggli, E. Regulatory function of Na-Ca exchange in the heart: milestones and outlook. The Journal of membrane biology 168, 107-130 (1999). Cerca con Google

52 Ferrari, R., Ceconi, C. & Guardigli, G. Pathophysiological role of heart rate: from ischaemia to left ventricular dysfunction. Vol. 10 (2008). Cerca con Google

53 Akar, F. G. & O'Rourke, B. Mitochondria are sources of metabolic sink and arrhythmias. Pharmacol Ther 131, 287-294, doi:10.1016/j.pharmthera.2011.04.005 (2011). Cerca con Google

54 Brown, D. A. & O'Rourke, B. Cardiac mitochondria and arrhythmias. Cardiovasc Res 88, 241-249, doi:10.1093/cvr/cvq231 (2010). Cerca con Google

55 Kohlhaas, M. et al. Elevated cytosolic Na+ increases mitochondrial formation of reactive oxygen species in failing cardiac myocytes. Circulation 121, 1606-1613, doi:10.1161/circulationaha.109.914911 (2010). Cerca con Google

56 Rizzuto, R. Calcium mobilization from mitochondria in synaptic transmitter release. The Journal of cell biology 163, 441-443, doi:10.1083/jcb.200309111 (2003). Cerca con Google

57 Dorn, G. W., 2nd & Scorrano, L. Two close, too close: sarcoplasmic reticulum-mitochondrial crosstalk and cardiomyocyte fate. Circ Res 107, 689-699, doi:10.1161/circresaha.110.225714 (2010). Cerca con Google

58 Deluca, H. F. & Engstrom, G. W. Calcium uptake by rat kidney mitochondria. Proceedings of the National Academy of Sciences of the United States of America 47, 1744-1750 (1961). Cerca con Google

59 Pradhan, R. K., Qi, F., Beard, D. A. & Dash, R. K. Characterization of membrane potential dependency of mitochondrial Ca2+ uptake by an improved biophysical model of mitochondrial Ca2+ uniporter. PLoS One 5, e13278, doi:10.1371/journal.pone.0013278 (2010). Cerca con Google

60 Kamer, K. J. & Mootha, V. K. The molecular era of the mitochondrial calcium uniporter. Nature Reviews Molecular Cell Biology 16, 545-553, doi:10.1038/nrm4039 (2015). Cerca con Google

61 Perocchi, F. et al. MICU1 encodes a mitochondrial EF hand protein required for Ca2+ uptake. Nature 467, 291-296, doi:10.1038/nature09358 (2010). Cerca con Google

62 Mallilankaraman, K. et al. MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism. Nature Cell Biology 14, 1336-1343, doi:10.1038/ncb2622 (2012). Cerca con Google

63 Plovanich, M. et al. MICU2, a paralog of MICU1, resides within the mitochondrial uniporter complex to regulate calcium handling. PLoS One 8, e55785, doi:10.1371/journal.pone.0055785 (2013). Cerca con Google

64 Sancak, Y. et al. EMRE Is an Essential Component of the Mitochondrial Calcium Uniporter Complex. Science 342, 1379-1382, doi:10.1126/science.1242993 (2013). Cerca con Google

65 Raffaello, A. et al. The mitochondrial calcium uniporter is a multimer that can include a dominant-negative pore-forming subunit. The EMBO Journal 32, 2362-2376, doi:10.1038/emboj.2013.157 (2013). Cerca con Google

66 Hoffman, N. E. et al. SLC25A23 augments mitochondrial Ca(2)(+) uptake, interacts with MCU, and induces oxidative stress-mediated cell death. Molecular biology of the cell 25, 936-947, doi:10.1091/mbc.E13-08-0502 (2014). Cerca con Google

67 Marchi, S. & Pinton, P. The mitochondrial calcium uniporter complex: molecular components, structure and physiopathological implications. J Physiol 592, 829-839, doi:10.1113/jphysiol.2013.268235 (2014). Cerca con Google

68 Lee, Y. et al. Structure and function of the N-terminal domain of the human mitochondrial calcium uniporter. EMBO reports 16, 1318-1333, doi:10.15252/embr.201540436 (2015). Cerca con Google

69 Oxenoid, K. et al. Architecture of the mitochondrial calcium uniporter. Nature 533, 269-273, doi:10.1038/nature17656 (2016). Cerca con Google

70 Patron, M. et al. MICU1 and MICU2 Finely Tune the Mitochondrial Ca2+ Uniporter by Exerting Opposite Effects on MCU Activity. Molecular Cell 53, 726-737, doi:10.1016/j.molcel.2014.01.013 (2014). Cerca con Google

71 Vais, H. et al. EMRE Is a Matrix Ca(2+) Sensor that Governs Gatekeeping of the Mitochondrial Ca(2+) Uniporter. Cell Rep 14, 403-410, doi:10.1016/j.celrep.2015.12.054 (2016). Cerca con Google

72 Tomar, D. et al. MCUR1 Is a Scaffold Factor for the MCU Complex Function and Promotes Mitochondrial Bioenergetics. Cell Reports 15, 1673-1685, doi:10.1016/j.celrep.2016.04.050 (2016). Cerca con Google

73 Paupe, V., Prudent, J., Dassa, Emmanuel P., Rendon, Olga Z. & Shoubridge, Eric A. CCDC90A (MCUR1) Is a Cytochrome c Oxidase Assembly Factor and Not a Regulator of the Mitochondrial Calcium Uniporter. Cell Metabolism 21, 109-116, doi:10.1016/j.cmet.2014.12.004 (2015). Cerca con Google

74 Fieni, F., Lee, S. B., Jan, Y. N. & Kirichok, Y. Activity of the mitochondrial calcium uniporter varies greatly between tissues. Nature communications 3, 1317-1317, doi:10.1038/ncomms2325 (2012). Cerca con Google

75 Holmström, K. M. et al. Assessment of cardiac function in mice lacking the mitochondrial calcium uniporter. Journal of Molecular and Cellular Cardiology 85, 178-182, doi:10.1016/j.yjmcc.2015.05.022 (2015). Cerca con Google

76 Oka, T. Cardiac-Specific Deletion of Gata4 Reveals Its Requirement for Hypertrophy, Compensation, and Myocyte Viability. Circulation Research 98, 837-845, doi:10.1161/01.RES.0000215985.18538.c4 (2006). Cerca con Google

77 Antony, A. N. et al. MICU1 regulation of mitochondrial Ca2+ uptake dictates survival and tissue regeneration. Nature Communications 7, 10955, doi:10.1038/ncomms10955 (2016). Cerca con Google

78 Liu, J. C. et al. MICU1 Serves as a Molecular Gatekeeper to Prevent In Vivo Mitochondrial Calcium Overload. Cell Reports 16, 1561-1573, doi:10.1016/j.celrep.2016.07.011 (2016). Cerca con Google

79 Logan, C. V. et al. Loss-of-function mutations in MICU1 cause a brain and muscle disorder linked to primary alterations in mitochondrial calcium signaling. Nature Genetics 46, 188-193, doi:10.1038/ng.2851 (2013). Cerca con Google

80 Bohovych, I. & Khalimonchuk, O. Sending Out an SOS: Mitochondria as a Signaling Hub. Frontiers in Cell and Developmental Biology 4, doi:10.3389/fcell.2016.00109 (2016). Cerca con Google

81 E., L. G., Stefano, S., David, S., Paul, B. & Margaret, B. Developmental regulation of myosin gene expression in mouse cardiac muscle. The Journal of cell biology 111, 2427-2436 (1990). Cerca con Google

82 Bass, A., Stejskalová, M., Stieglerová, A., Ostádal, B. & Samánek, M. Ontogenetic development of energy-supplying enzymes in rat and guinea-pig heart. Physiol Res 50, 237-245 (2001). Cerca con Google

83 Piquereau, J. et al. Postnatal development of mouse heart: formation of energetic microdomains. The Journal of Physiology 588, 2443-2454, doi:10.1113/jphysiol.2010.189670 (2010). Cerca con Google

84 Hoerter, J. A., Kuznetsov, A. & Ventura-Clapier, R. Functional development of the creatine kinase system in perinatal rabbit heart. Circ Res 69, 665-676 (1991). Cerca con Google

85 Seki, S. Fetal and postnatal development of Ca2+ transients and Ca2+ sparks in rat cardiomyocytes. Cardiovascular Research 58, 535-548, doi:10.1016/s0008-6363(03)00255-4 (2003). Cerca con Google

86 Carafoli, E., Garcia-Martin, E. & Guerini, D. The plasma membrane calcium pump: recent developments and future perspectives. Experientia 52, 1091-1100 (1996). Cerca con Google

87 Carafoli, E. Calcium pump of the plasma membrane. Physiol Rev 71, 129-153 (1991). Cerca con Google

88 Eghbali, M. et al. Molecular and functional signature of heart hypertrophy during pregnancy. Circ Res 96, 1208-1216, doi:10.1161/01.res.0000170652.71414.16 (2005). Cerca con Google

89 Winsor, T. & Beckner, G. Hypertrophy of the heart; electrocardiographic distinction between physiologic and pathologic enlargement. California medicine 82, 151-158 (1955). Cerca con Google

90 Maillet, M., van Berlo, J. H. & Molkentin, J. D. Molecular basis of physiological heart growth: fundamental concepts and new players. Nature Reviews Molecular Cell Biology 14, 38-48, doi:10.1038/nrm3495 (2012). Cerca con Google

91 Sugishita, Y., Koseki, S., Matsuda, M., Yamaguchi, T. & Ito, I. Myocardial mechanics of athletic hearts in comparison with diseased hearts. Am Heart J 105, 273-280 (1983). Cerca con Google

92 Bernardo, B. C., Weeks, K. L., Pretorius, L. & McMullen, J. R. Molecular distinction between physiological and pathological cardiac hypertrophy: Experimental findings and therapeutic strategies. Pharmacology & Therapeutics 128, 191-227, doi:10.1016/j.pharmthera.2010.04.005 (2010). Cerca con Google

93 Pluim, B. M., Zwinderman, A. H., van der Laarse, A. & van der Wall, E. E. The athlete's heart. A meta-analysis of cardiac structure and function. Circulation 101, 336-344 (2000). Cerca con Google

94 Sarquella-Brugada, G. et al. Genetics of sudden cardiac death in children and young athletes. Cardiology in the Young 23, 159-173, doi:10.1017/s1047951112001138 (2012). Cerca con Google

95 Patten, I. S. et al. Cardiac angiogenic imbalance leads to peripartum cardiomyopathy. Nature 485, 333-338, doi:10.1038/nature11040 (2012). Cerca con Google

96 Laughlin, M. H., Bowles, D. K. & Duncker, D. J. The coronary circulation in exercise training. American Journal of Physiology - Heart and Circulatory Physiology 302, H10-H23, doi:10.1152/ajpheart.00574.2011 (2012). Cerca con Google

97 Abel, E. D. & Doenst, T. Mitochondrial adaptations to physiological vs. pathological cardiac hypertrophy. Cardiovascular Research 90, 234-242, doi:10.1093/cvr/cvr015 (2011). Cerca con Google

98 Gertz, E. W., Wisneski, J. A., Stanley, W. C. & Neese, R. A. Myocardial substrate utilization during exercise in humans. Dual carbon-labeled carbohydrate isotope experiments. J Clin Invest 82, 2017-2025, doi:10.1172/jci113822 (1988). Cerca con Google

99 Braz, J. C. et al. Targeted inhibition of p38 MAPK promotes hypertrophic cardiomyopathy through upregulation of calcineurin-NFAT signaling. J Clin Invest 111, 1475-1486, doi:10.1172/jci17295 (2003). Cerca con Google

100 Aoyagi, T. & Matsui, T. Phosphoinositide-3 kinase signaling in cardiac hypertrophy and heart failure. Current pharmaceutical design 17, 1818-1824 (2011). Cerca con Google

101 Soesanto, W. et al. mTOR is a critical regulator of cardiac hypertrophy in Spontaneously Hypertensive Rats. Hypertension 54, 1321-1327, doi:10.1161/HYPERTENSIONAHA.109.138818 (2009). Cerca con Google

102 Boström, P. et al. C/EBPβ controls exercise-induced cardiac growth and protects against pathological cardiac remodeling. Cell 143, 1072-1083, doi:10.1016/j.cell.2010.11.036 (2010). Cerca con Google

103 Kehat, I. et al. Extracellular Signal-Regulated Kinases 1 and 2 Regulate the Balance Between Eccentric and Concentric Cardiac Growth. Circulation Research 108, 176-183, doi:10.1161/circresaha.110.231514 (2010). Cerca con Google

104 Kehat, I. & Molkentin, J. D. Extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in cardiac hypertrophy. Ann N Y Acad Sci 1188, 96-102, doi:10.1111/j.1749-6632.2009.05088.x (2010). Cerca con Google

105 Feldman, M. D. et al. Deficient production of cyclic AMP: pharmacologic evidence of an important cause of contractile dysfunction in patients with end-stage heart failure. Circulation 75, 331-339 (1987). Cerca con Google

106 Hasenfuss, G. Alterations of calcium-regulatory proteins in heart failure. Cardiovasc Res 37, 279-289 (1998). Cerca con Google

107 Schwinger, R. H. et al. Reduced Ca(2+)-sensitivity of SERCA 2a in failing human myocardium due to reduced serin-16 phospholamban phosphorylation. J Mol Cell Cardiol 31, 479-491 (1999). Cerca con Google

108 Respress, J. L. et al. Long-term simulated microgravity causes cardiac RyR2 phosphorylation and arrhythmias in mice. International Journal of Cardiology 176, 994-1000, doi:10.1016/j.ijcard.2014.08.138 (2014). Cerca con Google

109 Dobrev, D. & Wehrens, X. H. T. Role of RyR2 Phosphorylation in Heart Failure and Arrhythmias: Controversies Around Ryanodine Receptor Phosphorylation in Cardiac Disease. Circulation Research 114, 1311-1319, doi:10.1161/circresaha.114.300568 (2014). Cerca con Google

110 Houser, S. R. Role of RyR2 Phosphorylation in Heart Failure and Arrhythmias: Protein Kinase A-Mediated Hyperphosphorylation of the Ryanodine Receptor at Serine 2808 Does Not Alter Cardiac Contractility or Cause Heart Failure and Arrhythmias. Circulation Research 114, 1320-1327, doi:10.1161/circresaha.114.300569 (2014). Cerca con Google

111 Luo, M. & Anderson, M. E. Mechanisms of Altered Ca2+Handling in Heart Failure. Circulation Research 113, 690-708, doi:10.1161/circresaha.113.301651 (2013). Cerca con Google

112 Crossman, D. J., Ruygrok, P. N., Soeller, C. & Cannell, M. B. Changes in the organization of excitation-contraction coupling structures in failing human heart. PLoS One 6, e17901, doi:10.1371/journal.pone.0017901 (2011). Cerca con Google

113 Taegtmeyer, H. Switching Metabolic Genes to Build a Better Heart. Circulation 106, 2043-2045, doi:10.1161/01.cir.0000036760.42319.3f (2002). Cerca con Google

114 Akki, A., Smith, K. & Seymour, A.-M. L. Compensated cardiac hypertrophy is characterised by a decline in palmitate oxidation. Molecular and Cellular Biochemistry 311, 215-224, doi:10.1007/s11010-008-9711-y (2008). Cerca con Google

115 Lydell, C. P. et al. Pyruvate dehydrogenase and the regulation of glucose oxidation in hypertrophied rat hearts. Cardiovasc Res 53, 841-851 (2002). Cerca con Google

116 Friehs, I. et al. Vascular endothelial growth factor delays onset of failure in pressure–overload hypertrophy through matrix metalloproteinase activation and angiogenesis. Basic Research in Cardiology 101, 204-213, doi:10.1007/s00395-005-0581-0 (2005). Cerca con Google

117 Fowler, E. D. et al. Decreased creatine kinase is linked to diastolic dysfunction in rats with right heart failure induced by pulmonary artery hypertension. Journal of Molecular and Cellular Cardiology 86, 1-8, doi:10.1016/j.yjmcc.2015.06.016 (2015). Cerca con Google

118 Shiojima, I. Disruption of coordinated cardiac hypertrophy and angiogenesis contributes to the transition to heart failure. Journal of Clinical Investigation 115, 2108-2118, doi:10.1172/jci24682 (2005). Cerca con Google

119 Izumiya, Y. et al. Vascular Endothelial Growth Factor Blockade Promotes the Transition From Compensatory Cardiac Hypertrophy to Failure in Response to Pressure Overload. Hypertension 47, 887-893, doi:10.1161/01.hyp.0000215207.54689.31 (2006). Cerca con Google

120 Brown, R. D., Ambler, S. K., Mitchell, M. D. & Long, C. S. THE CARDIAC FIBROBLAST: Therapeutic Target in Myocardial Remodeling and Failure. Annual Review of Pharmacology and Toxicology 45, 657-687, doi:10.1146/annurev.pharmtox.45.120403.095802 (2005). Cerca con Google

121 Berk, B. C., Fujiwara, K. & Lehoux, S. ECM remodeling in hypertensive heart disease. The Journal of Clinical Investigation 117, 568-575, doi:10.1172/JCI31044 (2007). Cerca con Google

122 Gonzalez, G. E. et al. Deletion of interleukin-6 prevents cardiac inflammation, fibrosis and dysfunction without affecting blood pressure in angiotensin II-high salt-induced hypertension. Journal of hypertension 33, 144-152, doi:10.1097/hjh.0000000000000358 (2015). Cerca con Google

123 Mann, D. L. The emerging role of innate immunity in the heart and vascular system: for whom the cell tolls. Circ Res 108, 1133-1145, doi:10.1161/circresaha.110.226936 (2011). Cerca con Google

124 Mann, D. L. Inflammatory mediators and the failing heart: past, present, and the foreseeable future. Circ Res 91, 988-998 (2002). Cerca con Google

125 Du, X.-J. Distinct Role of Adrenoceptor Subtypes in Cardiac Adaptation to Chronic Pressure Overload. Clinical and Experimental Pharmacology and Physiology 35, 355-360, doi:10.1111/j.1440-1681.2007.04871.x (2008). Cerca con Google

126 Engelhardt, S., Hein, L., Wiesmann, F. & Lohse, M. J. Progressive hypertrophy and heart failure in beta1-adrenergic receptor transgenic mice. Proceedings of the National Academy of Sciences of the United States of America 96, 7059-7064 (1999). Cerca con Google

127 Bristow, M. R. beta-adrenergic receptor blockade in chronic heart failure. Circulation 101, 558-569 (2000). Cerca con Google

128 Kiriazis, H. et al. Knockout of β1- and β2-adrenoceptors attenuates pressure overload-induced cardiac hypertrophy and fibrosis. British Journal of Pharmacology 153, 684-692, doi:10.1038/sj.bjp.0707622 (2008). Cerca con Google

129 Bowling, N. et al. Increased protein kinase C activity and expression of Ca2+-sensitive isoforms in the failing human heart. Circulation 99, 384-391 (1999). Cerca con Google

130 Braz, J. C. et al. PKC-α regulates cardiac contractility and propensity toward heart failure. Nature Medicine 10, 248-254, doi:10.1038/nm1000 (2004). Cerca con Google

131 Bowman, J. C. et al. Expression of protein kinase C beta in the heart causes hypertrophy in adult mice and sudden death in neonates. Journal of Clinical Investigation 100, 2189-2195, doi:10.1172/jci119755 (1997). Cerca con Google

132 Haq, S. et al. Differential activation of signal transduction pathways in human hearts with hypertrophy versus advanced heart failure. Circulation 103, 670-677 (2001). Cerca con Google

133 Sussman, M. A. et al. Prevention of cardiac hypertrophy in mice by calcineurin inhibition. Science 281, 1690-1693 (1998). Cerca con Google

134 Zhang, T. The deltaC Isoform of CaMKII Is Activated in Cardiac Hypertrophy and Induces Dilated Cardiomyopathy and Heart Failure. Circulation Research 92, 912-919, doi:10.1161/01.res.0000069686.31472.c5 (2003). Cerca con Google

135 Westenbrink, B. D. et al. Mitochondrial Reprogramming Induced by CaMKII Mediates Hypertrophy Decompensation. Circulation Research 116, e28-e39, doi:10.1161/circresaha.116.304682 (2015). Cerca con Google

136 Kreusser, M. M. et al. Cardiac CaM Kinase II Genes δ and γ Contribute to Adverse Remodeling but Redundantly Inhibit Calcineurin-Induced Myocardial HypertrophyCLINICAL PERSPECTIVE. Circulation 130, 1262-1273, doi:10.1161/circulationaha.114.006185 (2014). Cerca con Google

137 Tham, Y. K., Bernardo, B. C., Ooi, J. Y. Y., Weeks, K. L. & McMullen, J. R. Pathophysiology of cardiac hypertrophy and heart failure: signaling pathways and novel therapeutic targets. Archives of Toxicology 89, 1401-1438, doi:10.1007/s00204-015-1477-x (2015). Cerca con Google

138 McMullen, J. R. et al. Protective effects of exercise and phosphoinositide 3-kinase(p110 ) signaling in dilated and hypertrophic cardiomyopathy. Proceedings of the National Academy of Sciences 104, 612-617, doi:10.1073/pnas.0606663104 (2007). Cerca con Google

139 Lin, Z. et al. Pi3kcb Links Hippo-YAP and PI3K-AKT Signaling Pathways to Promote Cardiomyocyte Proliferation and Survival. Circulation Research 116, 35-45, doi:10.1161/circresaha.115.304457 (2014). Cerca con Google

140 Zsebo, K. et al. Long-Term Effects of AAV1/SERCA2a Gene Transfer in Patients With Severe Heart FailureNovelty and Significance. Circulation Research 114, 101-108, doi:10.1161/circresaha.113.302421 (2014). Cerca con Google

141 Raake, P. W. et al. G Protein-Coupled Receptor Kinase 2 Ablation in Cardiac Myocytes Before or After Myocardial Infarction Prevents Heart Failure. Circulation Research 103, 413-422, doi:10.1161/circresaha.107.168336 (2008). Cerca con Google

142 Aiello, L. P. Inhibition of PKC by Oral Administration of Ruboxistaurin Is Well Tolerated and Ameliorates Diabetes-Induced Retinal Hemodynamic Abnormalities in Patients. Investigative Ophthalmology & Visual Science 47, 86-92, doi:10.1167/iovs.05-0757 (2006). Cerca con Google

143 Santulli, G., Xie, W., Reiken, S. R. & Marks, A. R. Mitochondrial calcium overload is a key determinant in heart failure. Proceedings of the National Academy of Sciences 112, 11389-11394, doi:10.1073/pnas.1513047112 (2015). Cerca con Google

144 Kindo, M. et al. Pressure overload-induced mild cardiac hypertrophy reduces left ventricular transmural differences in mitochondrial respiratory chain activity and increases oxidative stress. Frontiers in Physiology 3, doi:10.3389/fphys.2012.00332 (2012). Cerca con Google

145 Rosca, M. G. & Hoppel, C. L. Mitochondria in heart failure. Cardiovascular Research 88, 40-50, doi:10.1093/cvr/cvq240 (2010). Cerca con Google

146 Rosca, M. G., Tandler, B. & Hoppel, C. L. Mitochondria in cardiac hypertrophy and heart failure. Journal of Molecular and Cellular Cardiology 55, 31-41, doi:10.1016/j.yjmcc.2012.09.002 (2013). Cerca con Google

147 Taegtmeyer, H. & Overturf, M. L. Effects of moderate hypertension on cardiac function and metabolism in the rabbit. Hypertension 11, 416-426 (1988). Cerca con Google

148 Taegtmeyer, H., Golfman, L., Sharma, S., Razeghi, P. & van Arsdall, M. Linking gene expression to function: metabolic flexibility in the normal and diseased heart. Ann N Y Acad Sci 1015, 202-213, doi:10.1196/annals.1302.017 (2004). Cerca con Google

149 Bishop, S. P. & Altschuld, R. A. Increased glycolytic metabolism in cardiac hypertrophy and congestive failure. The American journal of physiology 218, 153-159 (1970). Cerca con Google

150 Taegtmeyer, H., Sen, S. & Vela, D. Return to the fetal gene program. Annals of the New York Academy of Sciences 1188, 191-198, doi:10.1111/j.1749-6632.2009.05100.x (2010). Cerca con Google

151 Taegtmeyer, H., Sen, S. & Vela, D. Return to the fetal gene program: a suggested metabolic link to gene expression in the heart. Ann N Y Acad Sci 1188, 191-198, doi:10.1111/j.1749-6632.2009.05100.x (2010). Cerca con Google

152 Sambandam, N., Lopaschuk, G. D., Brownsey, R. W. & Allard, M. F. Energy metabolism in the hypertrophied heart. Heart Fail Rev 7, 161-173 (2002). Cerca con Google

153 Allard, M. F., Schonekess, B. O., Henning, S. L., English, D. R. & Lopaschuk, G. D. Contribution of oxidative metabolism and glycolysis to ATP production in hypertrophied hearts. The American journal of physiology 267, H742-750 (1994). Cerca con Google

154 Hue, L. & Taegtmeyer, H. The Randle cycle revisited: a new head for an old hat. American journal of physiology. Endocrinology and metabolism 297, E578-591, doi:10.1152/ajpendo.00093.2009 (2009). Cerca con Google

155 Sorokina, N. et al. Recruitment of compensatory pathways to sustain oxidative flux with reduced carnitine palmitoyltransferase I activity characterizes inefficiency in energy metabolism in hypertrophied hearts. Circulation 115, 2033-2041, doi:10.1161/circulationaha.106.668665 (2007). Cerca con Google

156 Martin, M. A. et al. Myocardial carnitine and carnitine palmitoyltransferase deficiencies in patients with severe heart failure. Biochimica et biophysica acta 1502, 330-336 (2000). Cerca con Google

157 Graham, B. H. et al. A mouse model for mitochondrial myopathy and cardiomyopathy resulting from a deficiency in the heart/muscle isoform of the adenine nucleotide translocator. Nat Genet 16, 226-234, doi:10.1038/ng0797-226 (1997). Cerca con Google

158 Walther, T. et al. Accelerated mitochondrial adenosine diphosphate/adenosine triphosphate transport improves hypertension-induced heart disease. Circulation 115, 333-344, doi:10.1161/circulationaha.106.643296 (2007). Cerca con Google

159 Ingwall, J. S. & Weiss, R. G. Is the failing heart energy starved? On using chemical energy to support cardiac function. Circ Res 95, 135-145, doi:10.1161/01.RES.0000137170.41939.d9 (2004). Cerca con Google

160 Neubauer, S. The failing heart--an engine out of fuel. The New England journal of medicine 356, 1140-1151, doi:10.1056/NEJMra063052 (2007). Cerca con Google

161 Santulli, G. e. in Advances in Experimental Medicine and Biology Vol. 982 (Springer International Publishing AG 2017, 2017). Cerca con Google

162 Wu, Z. et al. Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 98, 115-124, doi:10.1016/s0092-8674(00)80611-x (1999). Cerca con Google

163 Puigserver, P. et al. A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92, 829-839 (1998). Cerca con Google

164 Esterbauer, H., Oberkofler, H., Krempler, F. & Patsch, W. Human peroxisome proliferator activated receptor gamma coactivator 1 (PPARGC1) gene: cDNA sequence, genomic organization, chromosomal localization, and tissue expression. Genomics 62, 98-102, doi:10.1006/geno.1999.5977 (1999). Cerca con Google

165 Arany, Z. et al. Transverse aortic constriction leads to accelerated heart failure in mice lacking PPAR-gamma coactivator 1alpha. Proceedings of the National Academy of Sciences of the United States of America 103, 10086-10091, doi:10.1073/pnas.0603615103 (2006). Cerca con Google

166 Riehle, C. et al. PGC-1beta deficiency accelerates the transition to heart failure in pressure overload hypertrophy. Circ Res 109, 783-793, doi:10.1161/circresaha.111.243964 (2011). Cerca con Google

167 Garnier, A. et al. Depressed mitochondrial transcription factors and oxidative capacity in rat failing cardiac and skeletal muscles. J Physiol 551, 491-501, doi:10.1113/jphysiol.2003.045104 (2003). Cerca con Google

168 Osterholt, M., Nguyen, T. D., Schwarzer, M. & Doenst, T. Alterations in mitochondrial function in cardiac hypertrophy and heart failure. Heart Failure Reviews 18, 645-656, doi:10.1007/s10741-012-9346-7 (2012). Cerca con Google

169 Hirotani, S. et al. Involvement of nuclear factor-kappaB and apoptosis signal-regulating kinase 1 in G-protein-coupled receptor agonist-induced cardiomyocyte hypertrophy. Circulation 105, 509-515 (2002). Cerca con Google

170 Pimentel, D. R. et al. Reactive oxygen species mediate amplitude-dependent hypertrophic and apoptotic responses to mechanical stretch in cardiac myocytes. Circ Res 89, 453-460 (2001). Cerca con Google

171 Nakamura, K. et al. Inhibitory effects of antioxidants on neonatal rat cardiac myocyte hypertrophy induced by tumor necrosis factor-alpha and angiotensin II. Circulation 98, 794-799 (1998). Cerca con Google

172 Xie, Z. et al. Intracellular reactive oxygen species mediate the linkage of Na+/K+-ATPase to hypertrophy and its marker genes in cardiac myocytes. The Journal of biological chemistry 274, 19323-19328 (1999). Cerca con Google

173 Dhalla, A. K. & Singal, P. K. Antioxidant changes in hypertrophied and failing guinea pig hearts. The American journal of physiology 266, H1280-1285 (1994). Cerca con Google

174 Kuroda, J. et al. NADPH oxidase 4 (Nox4) is a major source of oxidative stress in the failing heart. Proceedings of the National Academy of Sciences of the United States of America 107, 15565-15570, doi:10.1073/pnas.1002178107 (2010). Cerca con Google

175 Ago, T. et al. Upregulation of Nox4 by hypertrophic stimuli promotes apoptosis and mitochondrial dysfunction in cardiac myocytes. Circ Res 106, 1253-1264, doi:10.1161/circresaha.109.213116 (2010). Cerca con Google

176 Bauersachs, J. Aldosterone antagonism in heart failure: improvement of cardiac remodelling, endothelial dysfunction and platelet activation. European journal of clinical investigation 34, 649-652, doi:10.1111/j.1365-2362.2004.01400.x (2004). Cerca con Google

177 Adiga, I. K. & Nair, R. R. Multiple signaling pathways coordinately mediate reactive oxygen species dependent cardiomyocyte hypertrophy. Cell biochemistry and function 26, 346-351, doi:10.1002/cbf.1449 (2008). Cerca con Google

178 Zhang, W., Elimban, V., Nijjar, M. S., Gupta, S. K. & Dhalla, N. S. Role of mitogen-activated protein kinase in cardiac hypertrophy and heart failure. Experimental & Clinical Cardiology 8, 173-183 (2003). Cerca con Google

179 Liang, Q. & Molkentin, J. D. Redefining the roles of p38 and JNK signaling in cardiac hypertrophy: dichotomy between cultured myocytes and animal models. J Mol Cell Cardiol 35, 1385-1394 (2003). Cerca con Google

180 Li, H. L. et al. Epigallocathechin-3 gallate inhibits cardiac hypertrophy through blocking reactive oxidative species-dependent and -independent signal pathways. Free radical biology & medicine 40, 1756-1775 (2006). Cerca con Google

181 Cai, J. et al. Crocetin protects against cardiac hypertrophy by blocking MEK-ERK1/2 signalling pathway. Journal of cellular and molecular medicine 13, 909-925, doi:10.1111/j.1582-4934.2008.00620.x (2009). Cerca con Google

182 Zhang, L., Zhang, Z., Guo, H. & Wang, Y. Na+/K+-ATPase-mediated signal transduction and Na+/K+-ATPase regulation. Fundamental & Clinical Pharmacology 22, 615-621, doi:10.1111/j.1472-8206.2008.00620.x (2008). Cerca con Google

183 Guo, J., Gertsberg, Z., Ozgen, N. & Steinberg, S. F. p66Shc links alpha1-adrenergic receptors to a reactive oxygen species-dependent AKT-FOXO3A phosphorylation pathway in cardiomyocytes. Circ Res 104, 660-669, doi:10.1161/circresaha.108.186288 (2009). Cerca con Google

184 Zou, X. J., Yang, L. & Yao, S. L. Propofol depresses angiotensin II-induced cardiomyocyte hypertrophy in vitro. Experimental biology and medicine (Maywood, N.J.) 233, 200-208, doi:10.3181/0707-rm-206 (2008). Cerca con Google

185 Frey, N., McKinsey, T. A. & Olson, E. N. Decoding calcium signals involved in cardiac growth and function. Nat Med 6, 1221-1227, doi:10.1038/81321 (2000). Cerca con Google

186 Izem-Meziane, M. et al. Catecholamine-induced cardiac mitochondrial dysfunction and mPTP opening: protective effect of curcumin. American journal of physiology. Heart and circulatory physiology 302, H665-674, doi:10.1152/ajpheart.00467.2011 (2012). Cerca con Google

187 Viola, H. M. & Hool, L. C. Targeting calcium and the mitochondria in prevention of pathology in the heart. Current drug targets 12, 748-760 (2011). Cerca con Google

188 Zaglia, T. et al. Content of mitochondrial calcium uniporter (MCU) in cardiomyocytes is regulated by microRNA-1 in physiologic and pathologic hypertrophy. Proceedings of the National Academy of Sciences, 201708772, doi:10.1073/pnas.1708772114 (2017). Cerca con Google

189 Marchi, S. et al. Downregulation of the Mitochondrial Calcium Uniporter by Cancer-Related miR-25. Current Biology 23, 58-63, doi:10.1016/j.cub.2012.11.026 (2013). Cerca con Google

190 Mitchelson, K. R. Roles of the canonical myomiRs miR-1, -133 and -206 in cell development and disease. World Journal of Biological Chemistry 6, 162, doi:10.4331/wjbc.v6.i3.162 (2015). Cerca con Google

191 Kertesz, M., Iovino, N., Unnerstall, U., Gaul, U. & Segal, E. The role of site accessibility in microRNA target recognition. Nature Genetics 39, 1278-1284, doi:10.1038/ng2135 (2007). Cerca con Google

192 Latronico, M. V. G., Catalucci, D. & Condorelli, G. Emerging Role of MicroRNAs in Cardiovascular Biology. Circulation Research 101, 1225-1236, doi:10.1161/circresaha.107.163147 (2007). Cerca con Google

193 Zhao, Y., Samal, E. & Srivastava, D. Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature 436, 214-220, doi:10.1038/nature03817 (2005). Cerca con Google

194 Callis, T. E. et al. MicroRNA-208a is a regulator of cardiac hypertrophy and conduction in mice. Journal of Clinical Investigation 119, 2772-2786, doi:10.1172/JCI36154 (2009). Cerca con Google

195 Callis, T. E. & Wang, D. Z. Taking microRNAs to heart. Trends Mol Med 14, 254-260, doi:10.1016/j.molmed.2008.03.006 (2008). Cerca con Google

196 Terentyev, D. et al. miR-1 Overexpression Enhances Ca2+ Release and Promotes Cardiac Arrhythmogenesis by Targeting PP2A Regulatory Subunit B56 alpha and Causing CaMKII-Dependent Hyperphosphorylation of RyR2. Circulation Research 104, 514-521, doi:10.1161/Circresaha.108.181651 (2009). Cerca con Google

197 Tritsch, E. et al. An SRF/miR-1 axis regulates NCX1 and Annexin A5 protein levels in the normal and failing heart. Cardiovascular Research 98, 372-380, doi:10.1093/cvr/cvt042 (2013). Cerca con Google

198 Lyons, G. E., Schiaffino, S., Sassoon, D., Barton, P. & Buckingham, M. Developmental regulation of myosin gene expression in mouse cardiac muscle. J Cell Biol 111, 2427-2436 (1990). Cerca con Google

199 Anmann, T. et al. Formation of highly organized intracellular structure and energy metabolism in cardiac muscle cells during postnatal development of rat heart. Bba-Bioenergetics 1837, 1350-1361, doi:10.1016/j.bbabio.2014.03.015 (2014). Cerca con Google

200 Piquereau, J. et al. Postnatal development of mouse heart: formation of energetic microdomains. J Physiol-London 588, 2443-2454, doi:10.1113/jphysiol.2010.189670 (2010). Cerca con Google

201 Ikeda, S. et al. MicroRNA-1 negatively regulates expression of the hypertrophy-associated calmodulin and Mef2a genes. Mol Cell Biol 29, 2193-2204, doi:10.1128/MCB.01222-08 [pii] (2009). Cerca con Google

202 Knezevic, I. et al. A novel cardiomyocyte-enriched microRNA, miR-378, targets insulin-like growth factor 1 receptor: implications in postnatal cardiac remodeling and cell survival. J Biol Chem 287, 12913-12926, doi:10.1074/jbc.M111.331751 [pii] (2012). Cerca con Google

203 Goss, R. J. Hypertrophy versus hyperplasia. Science. 153, 1615-1620. (1966). Cerca con Google

204 Nadal-Ginard, B., Kajstura, J., Leri, A. & Anversa, P. Myocyte death, growth, and regeneration in cardiac hypertrophy and failure. Circ Res. 92, 139-150. (2003). Cerca con Google

205 Rumyantsev, P. P. Interrelations of the proliferation and differentiation processes during cardiact myogenesis and regeneration. Int Rev Cytol. 51, 186-273. (1977). Cerca con Google

206 Tuomainen, T. & Tavi, P. The role of cardiac energy metabolism in cardiac hypertrophy and failure. Experimental Cell Research, doi:10.1016/j.yexcr.2017.03.052 (2017). Cerca con Google

207 Rockman, H. A. et al. Segregation of atrial-specific and inducible expression of an atrial natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy. Proc Natl Acad Sci U S A 88, 8277-8281 (1991). Cerca con Google

208 Lu, X. et al. Measuring Local Gradients of Intramitochondrial [Ca2+] in Cardiac Myocytes During Sarcoplasmic Reticulum Ca2+ReleaseNovelty and Significance. Circulation Research 112, 424-431, doi:10.1161/circresaha.111.300501 (2013). Cerca con Google

209 Robert, V. et al. Beat-to-beat oscillations of mitochondrial [Ca2+] in cardiac cells. Embo J. 20, 4998-5007. (2001). Cerca con Google

210 Drago, I., De Stefani, D., Rizzuto, R. & Pozzan, T. Mitochondrial Ca2+ uptake contributes to buffering cytoplasmic Ca2+ peaks in cardiomyocytes. Proc Natl Acad Sci U S A. 109, 12986-12991. doi: 12910.11073/pnas.1210718109. Epub 1210712012 Jul 1210718120. (2012). Cerca con Google

211 Ambardekar, A. V. & Buttrick, P. M. Reverse Remodeling With Left Ventricular Assist Devices: A Review of Clinical, Cellular, and Molecular Effects. Circulation: Heart Failure 4, 224-233, doi:10.1161/circheartfailure.110.959684 (2011). Cerca con Google

212 Wilkins, B. J. Calcineurin/NFAT Coupling Participates in Pathological, but not Physiological, Cardiac Hypertrophy. Circulation Research 94, 110-118, doi:10.1161/01.res.0000109415.17511.18 (2004). Cerca con Google

213 Zhang, W. et al. β-Adrenergic Receptor-PI3K Signaling Crosstalk in Mouse Heart: Elucidation of Immediate Downstream Signaling Cascades. PLoS ONE 6, e26581, doi:10.1371/journal.pone.0026581 (2011). Cerca con Google

214 Erickson, J. R. et al. A Dynamic Pathway for Calcium-Independent Activation of CaMKII by Methionine Oxidation. Cell 133, 462-474, doi:10.1016/j.cell.2008.02.048 (2008). Cerca con Google

215 Erickson, J. R. Mechanisms of CaMKII Activation in the Heart. Frontiers in Pharmacology 5, doi:10.3389/fphar.2014.00059 (2014). Cerca con Google

216 Yang, H. T. et al. The ryanodine receptor modulates the spontaneous beating rate of cardiomyocytes during development. Proceedings of the National Academy of Sciences of the United States of America 99, 9225-9230, doi:10.1073/pnas.142651999 (2002). Cerca con Google

217 Bish, L. T. et al. Adeno-associated virus (AAV) serotype 9 provides global cardiac gene transfer superior to AAV1, AAV6, AAV7, and AAV8 in the mouse and rat. Human gene therapy 19, 1359-1368, doi:10.1089/hum.2008.123 (2008). Cerca con Google

218 Rose, T., Goltstein, P. M., Portugues, R. & Griesbeck, O. Putting a finishing touch on GECIs. Front Mol Neurosci 7, 88, doi:10.3389/fnmol.2014.00088 (2014). Cerca con Google

219 Akerboom, J. et al. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Frontiers in molecular neuroscience 6, 2, doi:10.3389/fnmol.2013.00002 (2013). Cerca con Google

220 Kotlikoff, M. I. Genetically encoded Ca2+ indicators: using genetics and molecular design to understand complex physiology. J Physiol 578, 55-67, doi:10.1113/jphysiol.2006.120212 (2007). Cerca con Google

221 Miyawaki, A., Griesbeck, O., Heim, R. & Tsien, R. Y. Dynamic and quantitative Ca2+ measurements using improved cameleons. Proceedings of the National Academy of Sciences of the United States of America 96, 2135-2140 (1999). Cerca con Google

222 Heim, R. & Tsien, R. Y. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6, 178-182 (1996). Cerca con Google

223 McCombs, J. E. & Palmer, A. E. Measuring calcium dynamics in living cells with genetically encodable calcium indicators. Methods 46, 152-159, doi:10.1016/j.ymeth.2008.09.015 S1046-2023(08)00166-7 [pii] (2008). Cerca con Google

224 Griffiths, E. J., Balaska, D. & Cheng, W. H. The ups and downs of mitochondrial calcium signalling in the heart. Biochim Biophys Acta 1797, 856-864, doi:10.1016/j.bbabio.2010.02.022 (2010). Cerca con Google

225 O'Rourke, B. & Blatter, L. A. Mitochondrial Ca2+ uptake: tortoise or hare? J Mol Cell Cardiol 46, 767-774, doi:10.1016/j.yjmcc.2008.12.011 (2009). Cerca con Google

226 Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882-887, doi:10.1038/42264 (1997). Cerca con Google

227 Palmer, A. E. & Tsien, R. Y. Measuring calcium signaling using genetically targetable fluorescent indicators. Nat Protoc 1, 1057-1065, doi:10.1038/nprot.2006.172 (2006). Cerca con Google

228 Greotti, E., Wong, A., Pozzan, T., Pendin, D. & Pizzo, P. Characterization of the ER-Targeted Low Affinity Ca2+ Probe D4ER. Sensors 16, 1419, doi:10.3390/s16091419 (2016). Cerca con Google

229 Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M. & Miyawaki, A. Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins. Proceedings of the National Academy of Sciences of the United States of America 101, 10554-10559, doi:10.1073/pnas.0400417101 (2004). Cerca con Google

230 Akerboom, J. et al. Optimization of a GCaMP calcium indicator for neural activity imaging. The Journal of neuroscience : the official journal of the Society for Neuroscience 32, 13819-13840, doi:10.1523/jneurosci.2601-12.2012 (2012). Cerca con Google

231 Chen, T. W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295-300, doi:10.1038/nature12354 (2013). Cerca con Google

232 Hanson, G. T. et al. Investigating Mitochondrial Redox Potential with Redox-sensitive Green Fluorescent Protein Indicators. Journal of Biological Chemistry 279, 13044-13053, doi:10.1074/jbc.M312846200 (2004). Cerca con Google

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