Structure, activity regulation, and role of the mitochondrial calcium uniporter in health and disease

Mitochondrial Ca2+ uptake plays a pivotal role both in cell energy balance and in cell fate determination. Studies on the role of mitochondrial Ca2+ signaling in pathophysiology have been favored by the identification of the genes encoding the mitochondrial calcium uniporter (MCU) and its regulatory subunits. Thus, research carried on in the last years on one hand has determined the structure of the MCU complex and its regulation, on the other has uncovered the consequences of dysregulated mitochondrial Ca2+ signaling in cell and tissue homeostasis. Whether mitochondrial Ca2+ uptake can be exploited as a weapon to counteract cancer progression is debated. In this review, we summarize recent research on the molecular structure of the MCU, the regulatory mechanisms that control its activity and its relevance in pathophysiology, focusing in particular on its role in cancer progression

Gene expression changes of single skeletal muscle fibers in response to modulation of the mitochondrial calcium uniporter (MCU)

The mitochondrial calcium uniporter (MCU) gene codifies for the inner mitochondrial membrane (IMM) channel responsible for mitochondrial Ca2 + uptake. Cytosolic Ca2 + transients are involved in sarcomere contraction through cycles of release and storage in the sarcoplasmic reticulum. In addition cytosolic Ca2 + regulates various signaling cascades that eventually lead to gene expression reprogramming. Mitochondria are strategically placed in close contact with the ER/SR, thus cytosolic Ca2 + transients elicit large increases in the [Ca2 +] of the mitochondrial matrix ([Ca2 +]mt). Mitochondrial Ca2 + uptake regulates energy production and cell survival. In addition, we recently showed that MCU-dependent mitochondrial Ca2 + uptake controls skeletal muscle trophism. In the same report, we dissected the effects of MCU-dependent mitochondrial Ca2 + uptake on gene expression through microarray gene expression analysis upon modulation of MCU expression by in vivo AAV infection. Analyses were performed on single skeletal muscle fibers at two time points (7 and 14 days post-AAV injection). Raw and normalized data are available on the GEO database (http://www.ncbi.nlm.nih.gov/geo/) (GSE60931)

The physiopathological role of mitochondrial calcium uptake in skeletal muscle homeostasis

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

The physiopathological role of mitochondrial calcium uptake in skeletal muscle homeostasis

In a wide variety of cell types, cytosolic Ca2+ transients, generated by physiological stimuli, elicit large increases in the [Ca2+] of the mitochondrial matrix, which in turn stimulate the Ca2+-sensitive dehydrogenases of the Krebs cycle. Rapid uptake is favored by the close proximity with the major Ca2+ store of the cell, namely the endoplasmic/sarcoplasmic reticulum (ER/SR), and thus by the exposure to high [Ca2+] microdomains. In addition, mitochondrial Ca2+ could contribute to the cellular homeostasis thanks to the existence of a sophisticated machinery, that allows this organelle to rapidly change its Ca2+ concentration (Rizzuto et al., 2012). This general picture is also apparent in skeletal muscle during contraction whereby agonist stimulation induces high amplitude mitochondrial Ca2+ increases in vivo (Rudolf et al., 2004), thus acting as buffers of the cytosolic [Ca2+] increase. Finally, mitochondrial Ca2+ stimulates aerobic metabolism and ATP production, that are essential for muscle activity. Indeed, mitochondria are the major source of ATP in oxidative fibres. However, excessive Ca2+ accumulation in mitochondria, a condition known as mitochondrial Ca2+ overload, can trigger cell death. The recent molecular identification of the Mitochondrial Calcium Uniporter (MCU), the highly selective channel responsible for Ca2+ entry into mitochondria, allows the detailed investigation of its role in different aspects of skeletal muscle biology (De Stefani et al., 2011; Baughman et al., 2011). The major goal of my PhD project was to address the role of mitochondrial Ca2+ in skeletal muscle homeostasis. For this purpose, we firstly investigated in vivo the effects of mitochondrial Ca2+ homeostasis in skeletal muscle function by overexpressing or silencing MCU by means of AAV vectors. We demonstrated that the modulation of MCU protein controls skeletal muscle size during both post-natal growth and adulthood. In detail, we observed an increase in fibre size in MCU-infected muscles. Conversely, MCU-silenced muscles displayed an atrophic phenotype. These striking phenomenon impinges on two major hypertrophic pathways, i.e. PGC-1α4 and IGF1-AKT. We thus explored two potential different mechanisms that could account for the MCU-dependent control of anabolic pathways, i) the activation of a mitochondria-to-nucleus signaling route, ii) the regulation of metabolites as signaling molecules. Regarding the mitochondria-to-nucleus route, we carried out a study on the PGC-1α4 promoter activity, and we demonstrated that mitochondrial Ca2+ controls the promoter activity of PGC-1α4. Concerning the involvement of cellular metabolism, we carried out steady-state metabolomics analyses of MCU-overexpressing and MCU-silencing muscles. We discovered a marked metabolic reprogramming in silenced muscles, including a clear shift from glucose metabolism toward preferential fatty acid β-oxidation. Next, we generated a skeletal muscle specific mcu knockout mouse (mlc1f-Cre-mcu-/-), by crossing a mcu fl/fl mouse with a line expressing the Cre recombinase under the control of the myosin light chain 1f (mlc1f) promoter. We observed marginal difference in fibre size of mlc1f-Cre-mcu-/- skeletal muscles. However, when these mice were exercised on a treadmill using different training protocols, an impaired running capacity of mlc1f-Cre-mcu-/- became evident, indicating that mitochondrial Ca2+ accumulation is required to guarantee skeletal muscle performance. Finally, it is well-established that Ca2+ plays a pivotal role in autophagy regulation. Thus, we decided to investigate this process in MCU-overexpressing and MCU-silencing muscles. We demonstrated that mitochondrial Ca2+ uptake modulation controls mitophagy without affecting bulk autophagy. Taken together, these data indicate that mitochondrial Ca2+ uptake plays a pivotal role in the control of skeletal muscle trophism. Further investigations of MCU-dependent effects on skeletal muscle homeostasis represent an important task for the future. Indeed, this research will provide new possible targets for clinical intervention in all diseases characterized by muscle loss, such as dystrophies, cancer cachexia and aging

Calcium at the Center of Cell Signaling: Interplay between Endoplasmic Reticulum, Mitochondria, and Lysosomes

In recent years, rapid discoveries have been made relating to Ca2+ handling at specific organelles that have important implications for whole-cell Ca2+ homeostasis. In particular, the structures of the endoplasmic reticulum (ER) Ca2+ channels revealed by electron cryomicroscopy (cryo-EM), continuous updates on the structure, regulation, and role of the mitochondrial calcium uniporter (MCU) complex, and the analysis of lysosomal Ca2+ signaling are milestones on the route towards a deeper comprehension of the complexity of global Ca2+ signaling. In this review we summarize recent discoveries on the regulation of interorganellar Ca2+ homeostasis and its role in pathophysiology

CoQ10 and Resveratrol Effects to Ameliorate Aged-Related Mitochondrial Dysfunctions

Mitochondria participate in the maintenance of cellular homeostasis. Firstly, mitochondria regulate energy metabolism through oxidative phosphorylation. In addition, they are involved in cell fate decisions by activating the apoptotic intrinsic pathway. Finally, they work as intracellular signaling hubs as a result of their tight regulation of ion and metabolite concentrations and other critical signaling molecules such as ROS. Aging is a multifactorial process triggered by impairments in different cellular components. Among the various molecular pathways involved, mitochondria are key regulators of longevity. Indeed, mitochondrial deterioration is a critical signature of the aging process. In this scenario, we will focus specifically on the age-related decrease in CoQ levels, an essential component of the electron transport chain (ETC) and an antioxidant, and how CoQ supplementation could benefit the aging process. Generally, any treatment that improves and sustains mitochondrial functionality is a good candidate to counteract age-related mitochondrial dysfunctions. In recent years, heightened attention has been given to natural compounds that modulate mitochondrial function. One of the most famous is resveratrol due to its ability to increase mitochondrial biogenesis and work as an antioxidant agent. This review will discuss recent clinical trials and meta-analyses based on resveratrol and CoQ supplementation, focusing on how these compounds could improve mitochondrial functionality during aging

Myonecrosis Induction by Intramuscular Injection of CTX

Skeletal muscle, one of the most abundant tissue in the body, is a highly regenerative tissue. Indeed, compared to other tissues that are not able to regenerate after injury, skeletal muscle can fully regenerate upon mechanically, chemically, and infection-induced trauma. Several injury models have been developed to thoroughly investigate the physiological mechanisms regulating skeletal muscle regeneration. This protocol describes how to induce muscle regeneration by taking advantage of a cardiotoxin (CTX)-induced muscle injury model. The overall steps include CTX injection of tibialis anterior (TA) muscles of BL6N mice, collection of regenerating muscles at different time points after CTX injury, and histological characterization of regenerating muscles. Our protocol, compared with others such as those for freeze-induced injury models, avoids laceration or infections of the muscles since it involves neither surgery nor suture. In addition, our protocol is highly reproducible, since it causes homogenous myonecrosis of the whole muscle, and further reduces animal pain and stress