OPA1 disease-causing mutants have domain-specific effects on mitochondrial ultrastructure and fusion.

Inner mitochondrial membrane fusion and cristae shape depend on optic atrophy protein 1, OPA1. Mutations in OPA1 lead to autosomal dominant optic atrophy (ADOA), an important cause of inherited blindness. The Guanosin Triphosphatase (GTPase) and GTPase effector domains (GEDs) of OPA1 are essential for mitochondrial fusion; yet, their specific roles remain elusive. Intriguingly, patients carrying OPA1 GTPase mutations have a higher risk of developing more severe multisystemic symptoms in addition to optic atrophy, suggesting pathogenic contributions for the GTPase and GED domains, respectively. We studied OPA1 GTPase and GED mutations to understand their domain-specific contribution to protein function by analyzing patient-derived cells and gain-of-function paradigms. Mitochondria from OPA1 GTPase (c.870+5G>A and c.889C>T) and GED (c.2713C>T and c.2818+5G>A) mutants display distinct aberrant cristae ultrastructure. While all OPA1 mutants inhibited mitochondrial fusion, some GTPase mutants resulted in elongated mitochondria, suggesting fission inhibition. We show that the GED is dispensable for fusion and OPA1 oligomer formation but necessary for GTPase activity. Finally, splicing defect mutants displayed a posttranslational haploinsufficiency-like phenotype but retained domain-specific dysfunctions. Thus, OPA1 domain-specific mutants result in distinct impairments in mitochondrial dynamics, providing insight into OPA1 function and its contribution to ADOA pathogenesis and severity.

Mitochondrial function in spinal cord injury and regeneration.

Many people around the world suffer from some form of paralysis caused by spinal cord injury (SCI), which has an impact on quality and life expectancy. The spinal cord is part of the central nervous system (CNS), which in mammals is unable to regenerate, and to date, there is a lack of full functional recovery therapies for SCI. These injuries start with a rapid and mechanical insult, followed by a secondary phase leading progressively to greater damage. This secondary phase can be potentially modifiable through targeted therapies. The growing literature, derived from mammalian and regenerative model studies, supports a leading role for mitochondria in every cellular response after SCI: mitochondrial dysfunction is the common event of different triggers leading to cell death, cellular metabolism regulates the immune response, mitochondrial number and localization correlate with axon regenerative capacity, while mitochondrial abundance and substrate utilization regulate neural stem progenitor cells self-renewal and differentiation. Herein, we present a comprehensive review of the cellular responses during the secondary phase of SCI, the mitochondrial contribution to each of them, as well as evidence of mitochondrial involvement in spinal cord regeneration, suggesting that a more in-depth study of mitochondrial function and regulation is needed to identify potential targets for SCI therapeutic intervention.

OPA1 Modulates Mitochondrial Ca2+ Uptake Through ER-Mitochondria Coupling.

Autosomal Dominant Optic Atrophy (ADOA), a disease that causes blindness and other neurological disorders, is linked to OPA1 mutations. OPA1, dependent on its GTPase and GED domains, governs inner mitochondrial membrane (IMM) fusion and cristae organization, which are central to oxidative metabolism. Mitochondrial dynamics and IMM organization have also been implicated in Ca2+ homeostasis and signaling but the specific involvements of OPA1 in Ca2+ dynamics remain to be established. Here we studied the possible outcomes of OPA1 and its ADOA-linked mutations in Ca2+ homeostasis using rescue and overexpression strategies in Opa1-deficient and wild-type murine embryonic fibroblasts (MEFs), respectively and in human ADOA-derived fibroblasts. MEFs lacking Opa1 required less Ca2+ mobilization from the endoplasmic reticulum (ER) to induce a mitochondrial matrix [Ca2+] rise ([Ca2+]mito). This was associated with closer ER-mitochondria contacts and no significant changes in the mitochondrial calcium uniporter complex. Patient cells carrying OPA1 GTPase or GED domain mutations also exhibited altered Ca2+ homeostasis, and the mutations associated with lower OPA1 levels displayed closer ER-mitochondria gaps. Furthermore, in Opa1 -/- MEF background, we found that acute expression of OPA1 GTPase mutants but no GED mutants, partially restored cytosolic [Ca2+] ([Ca2+]cyto) needed for a prompt [Ca2+]mito rise. Finally, OPA1 mutants' overexpression in WT MEFs disrupted Ca2+ homeostasis, partially recapitulating the observations in ADOA patient cells. Thus, OPA1 modulates functional ER-mitochondria coupling likely through the OPA1 GED domain in Opa1 -/- MEFs. However, the co-existence of WT and mutant forms of OPA1 in patients promotes an imbalance of Ca2+ homeostasis without a domain-specific effect, likely contributing to the overall ADOA progress.

ß-Hydroxybutyrate Increases Exercise Capacity Associated with Changes in Mitochondrial Function in Skeletal Muscle.

ß-hydroxybutyrate is the main ketone body generated by the liver under starvation. Under these conditions, it can sustain ATP levels by its oxidation in mitochondria. As mitochondria can modify its shape and function under different nutritional challenges, we study the chronic effects of ß-hydroxybutyrate supplementation on mitochondrial morphology and function, and its relation to exercise capacity. Male C57BL/6 mice were supplemented with ß-hydroxybutyrate mineral salt (3.2%) or control (CT, NaCl/KCl) for six weeks and submitted to a weekly exercise performance test. We found an increase in distance, maximal speed, and time to exhaustion at two weeks of supplementation. Fatty acid metabolism and OXPHOS subunit proteins declined at two weeks in soleus but not in tibialis anterior muscles. Oxygen consumption rate on permeabilized fibers indicated a decrease in the presence of pyruvate in the short-term treatment. Both the tibialis anterior and soleus showed decreased levels of Mitofusin 2, while electron microscopy assessment revealed a significant reduction in mitochondrial cristae shape in the tibialis anterior, while a reduction in the mitochondrial number was observed only in soleus. These results suggest that short, but not long-term, ß­hydroxybutyrate supplementation increases exercise capacity, associated with modifications in mitochondrial morphology and function in mouse skeletal muscle.

Inhibition of chymotrypsin-like activity of the proteasome by ixazomib prevents mitochondrial dysfunction during myocardial ischemia.

The heart is critically dependent on mitochondrial respiration for energy supply. Ischemia decreases oxygen availability, with catastrophic consequences for cellular energy systems. After a few minutes of ischemia, the mitochondrial respiratory chain halts, ATP levels drop and ion gradients across cell membranes collapse. Activation of cellular proteases and generation of reactive oxygen species by mitochondria during ischemia alter mitochondrial membrane permeability, causing mitochondrial swelling and fragmentation and eventually cell death. The mitochondria, therefore, are important targets of cardioprotection against ischemic injury. We have previously shown that ixazomib (IXA), a proteasome inhibitor used for treating multiple myeloma, effectively reduced the size of the infarct produced by global ischemia in isolated rat hearts and prevented degradation of the sarcoplasmic reticulum calcium release channel RyR2. The aim of this work was to further characterize the protective effect of IXA by determining its effect on mitochondrial morphology and function after ischemia. We also quantified the effect of IXA on levels of mitofusin-2, a protein involved in maintaining mitochondrial morphology and mitochondria-SR communication. We found that mitochondria were significantly preserved and functional parameters such as oxygen consumption, the ability to generate a membrane potential, and glutathione content were improved in mitochondria isolated from hearts perfused with IXA prior to ischemia. IXA also blocked the release of cytochrome c observed in ischemia and significantly preserved mitofusin-2 integrity. These beneficial effects resulted in a significant decrease in the left ventricular end diastolic pressure upon reperfusion and a smaller infarct in isolated hearts.

Skeletal muscle excitation-metabolism coupling.

Mitochondria represent the main source of ATP in skeletal muscle and mitochondria activity increases after muscle fiber depolarization. The regulation of mitochondrial function during contraction in skeletal muscle, however, is poorly understood. Skeletal muscle has a particular distribution of mitochondria where three distinct populations can be recognized. The most studied populations are the ones positioned deep into the myofibers between the myofibrils (intermyofibrillar mitochondria), and that located immediately beneath sarcolemma (subsarcolemmal mitochondria); a less studied population locates covering the myonuclei, as a continuation of the subsarcolemmal population. All mitochondria populations undergo fusion and fission events and intermyofibrillar mitochondria are interconnected; mitochondrial communication is necessary to maintain not only the energetic homeostasis of the muscle but its contractile function, as well. The mechanism supporting communication between subsarcolemmal and intermyofibrillar mitochondria is unknown. The recently described MCU complex of proteins has provided a new insight into the role of calcium as a regulator of mitochondrial function. Whether the different mitochondria populations have different calcium handling capacity and whether mitochondria Ca2+ has a role in energy transmission along the mitochondria network are intriguing issues that emerge when studying the link between electrical stimulation of the muscle fiber and the mitochondria metabolic output.

Muscle function decline and mitochondria changes in middle age precede sarcopenia in mice.

Sarcopenia is the degenerative loss of muscle mass and strength with aging. Although a role of mitochondrial metabolism in muscle function and in the development of many diseases has been described, the role of mitochondrial topology and dynamics in the process of muscle aging is not fully understood. This work shows a time line of changes in both mitochondrial distribution and skeletal muscle function during mice lifespan. We isolated muscle fibers from flexor digitorum brevis of mice of different ages. A fusion-like phenotype of mitochondria, together with a change in orientation perpendicular to the fiber axis was evident in the Adult group compared to Juvenile and Older groups. Moreover, an increase in the contact area between sarcoplasmic reticulum and mitochondria was evident in the same group. Together with the morphological changes, mitochondrial Ca2+ resting levels were reduced at age 10-14 months and significantly increased in the Older group. This was consistent with a reduced number of mitochondria-to-jSR pairs in the Older group compared to the Juvenile. Our results support the idea of several age-dependent changes in mitochondria that are accentuated in midlife prior to a complete sarcopenic phenotype.

ATP release due to Thy-1-integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation.

Thy-1, an abundant mammalian glycoprotein, interacts with αvß3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Cα upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between αvß3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion.

Mitochondria fine-tune the slow Ca(2+) transients induced by electrical stimulation of skeletal myotubes.

Mitochondria sense cytoplasmic Ca(2+) signals in many cell types. In mammalian skeletal myotubes, depolarizing stimuli induce two independent cytoplasmic Ca(2+) signals: a fast signal associated with contraction and a slow signal that propagates to the nucleus and regulates gene expression. How mitochondria sense and possibly affect these cytoplasmic Ca(2+) signals has not been reported. We investigated here (a) the emergence of mitochondrial Ca(2+) signals in response to electrical stimulation of myotubes, (b) the contribution of mitochondrial Ca(2+) transients to ATP generation and (c) the influence of mitochondria as modulators of cytoplasmic and nuclear Ca(2+) signals. Rhod2 and Fluo3 fluorescence determinations revealed composite Ca(2+) signals associated to the mitochondrial compartment in electrically stimulated (400 pulses, 45 Hz) skeletal myotubes. Similar Ca(2+) signals were detected when using a mitochondria-targeted pericam. The fast mitochondrial Ca(2+) rise induced by stimulation was inhibited by pre-incubation with ryanodine, whereas the phospholipase C inhibitor U73122 blocked the slow mitochondrial Ca(2+) signal, showing that mitochondria sense the two cytoplasmic Ca(2+) signal components. The fast but not the slow Ca(2+) transient enhanced mitochondrial ATP production. Inhibition of the mitochondrial Ca(2+) uniporter prevented the emergence of mitochondrial Ca(2+) transients and significantly increased the magnitude of slow cytoplasmic Ca(2+) signals after stimulation. Precluding mitochondrial Ca(2+) extrusion with the Na(+)/Ca(2+) exchanger inhibitor CGP37157 decreased mitochondrial potential, increased the magnitude of the slow cytoplasmic Ca(2+) signal and decreased the rate of Ca(2+) signal propagation from one nucleus to the next. Over expression of the mitochondrial fission protein Drp-1 decreased mitochondrial size and the slow Ca(2+) transient in mitochondria, but enhanced cytoplasmic and nuclear slow transients. The present results indicate that mitochondria play a central role in the regulation of Ca(2+) signals involved in gene expression in myotubes.

Changes in mitochondrial dynamics during ceramide-induced cardiomyocyte early apoptosis.

AIMS: In cells, mitochondria are organized as a network of interconnected organelles that fluctuate between fission and fusion events (mitochondrial dynamics). This process is associated with cell death. We investigated whether activation of apoptosis with ceramides affects mitochondrial dynamics and promotes mitochondrial fission in cardiomyocytes. METHODS AND RESULTS: Neonatal rat cardiomyocytes were incubated with C(2)-ceramide or the inactive analog dihydro-C(2)-ceramide for up to 6 h. Three-dimensional images of cells loaded with mitotracker green were obtained by confocal microscopy. Dynamin-related protein-1 (Drp-1) and mitochondrial fission protein 1 (Fis1) distribution and levels were studied by immunofluorescence and western blot. Mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c (cyt c) distribution were used as indexes of early activation of apoptosis. Cell viability and DNA fragmentation were determined by propidium iodide staining/flow cytometry, whereas cytotoxicity was evaluated by lactic dehydrogenase activity. To decrease the levels of the mitochondrial fusion protein mitofusin 2, we used an antisense adenovirus (AsMfn2). C(2)-ceramide, but not dihydro-C(2)-ceramide, promoted rapid fragmentation of the mitochondrial network in a concentration- and time-dependent manner. C(2)-ceramide also increased mitochondrial Drp-1 and Fis1 content, Drp-1 colocalization with Fis1, and caused early activation of apoptosis. AsMfn2 accentuated the decrease in DeltaPsi(m) and cyt c redistribution induced by C(2)-ceramide. Doxorubicin, which induces cardiomyopathy and apoptosis through ceramide generation, also stimulated mitochondrial fragmentation. CONCLUSION: Ceramides stimulate mitochondrial fission and this event is associated with early activation of cardiomyocyte apoptosis.

Tumor necrosis factor-alpha activates nuclear factor-kappaB but does not regulate progesterone production in cultured human granulosa luteal cells.

BACKGROUND: The role of tumor necrosis factor-alpha (TNF-alpha) in granulosa luteal cell function and steroidogenesis is still controversial. Our aim was to examine the steroidogenic response, together with the simultaneous expression and activation of nuclear factor-kappaB (NF-kappaB), in cultured human granulosa luteal cells (GLCs) following administration of TNF-alpha. MATERIALS AND METHODS: This prospective controlled study was conducted in the Human Reproduction Division at the Institute of Maternal and Child Research, Faculty of Medicine, University of Chile and the San Borja Arriarán Hospital, National Health Service, Santiago, Chile. GLCs were obtained from aspirates of follicles from women undergoing in vitro fertilization (IVF). Thirty-two women undergoing IVF for tubal-factor and/or male-factor infertility participated in this study. Protein levels of NF-kappaB, the NF-kappaB inhibitor IkappaBalpha and steroidogenic acute regulatory protein (StAR) were determined by Western blot and localization of NF-kappaB was studied by indirect immunofluorescence. Progesterone production was determined by radioimmunoassay. RESULTS: TNF-alpha did not affect the expression of StAR protein or the synthesis of progesterone. NF-kappaB was expressed in the GLCs and activated by TNF-alpha, resulting in degradation of IkappaBalpha and mobilization of the p65 NF-kappaB subunit into the nucleus. CONCLUSIONS: These results indicate that TNF-alpha did not modulate steroidogenesis in cultured human GLCs. However, NF-kappaB was activated by TNF-alpha. Therefore the activation of NF-kappaB via the TNF-alpha pathway is likely associated with other preovulatory granulosa cell processes important for human ovarian function.

IGF-1: un factor de crecimiento con acciones cardiovasculares pleiotrópicas / IGF-1: a growth factor with pleiotropic cardiovascular actions

El factor de crecimiento análogo a insulina tipo 1 (IGF-1) es un péptido relacionado estructural y funcionalmente con insulina que posee efectos mitogénicos y citoprotectores. Sus efectos biológicos dependen de la activación del receptor de IGF- 1 (IGF-1R), perteneciente a la familia de receptores con actividad tirosina kinasa intrínseca y que se localiza en la superficie celular. IGF-1 es el principal mediador fisiológico de la hormona del crecimiento y dado que su gen se expresa en múltiplestejidos, este factor es clave en la comunicación endocrina, paracrina y autocrina. Recientes evidencias muestran que IGF- 1 ejerce variadas acciones pleiotrópicas en el sistema cardiovascular, destacándose sus efectos en la hipertrofia, muerte y regeneración celular. En el corazón, IGF-1 promueve su crecimiento, mejora su contractibilidad, facilita el metabolismode la glucosa, disminuye el nivel de insulina circulante, aumenta la sensibilidad a esta hormona, estabiliza el perfil lipídico y estimula la regeneración del músculo cardíaco. Evidencias clínicas y experimentales han mostrado que el deterioro de la función cardíaca se asocia a bajos niveles circulantes de IGF-1. Alteraciones tanto en los niveles de IGF-1 como en su sistema transduccional se consideran factores de riesgo para el desarrollo de distintas patologías cardíacas. Todosestos antecedentes destacan el papel del IGF-1 en cardioprotección y su potencialidad para el tratamiento de diversas patologías cardiovasculares. Sin embargo, los mecanismos moleculares implicados en estos efectos prácticamente se desconocen. En esta revisión, junto con entregar antecedentes actualizados y críticos de las acciones cardiovasculares del IGF-1, se proyectan sus aplicaciones terapéuticas.

Hyperosmotic stress-dependent NFkappaB activation is regulated by reactive oxygen species and IGF-1 in cultured cardiomyocytes.

We have recently shown that hyperosmotic stress activates p65/RelB NFkappaB in cultured cardiomyocytes with dichotomic actions on caspase activation and cell death. It remains unexplored how NFkappaB is regulated in cultured rat cardiomyocytes exposed to hyperosmotic stress. We study here: (a) if hyperosmotic stress triggers reactive oxygen species (ROS) generation and in turn whether they regulate NFkappaB and (b) if insulin-like growth factor-1 (IGF-1) modulates ROS production and NFkappaB activation in hyperosmotically-stressed cardiomyocytes. The results showed that hyperosmotic stress generated ROS in cultured cardiac myocytes, in particular the hydroxyl and superoxide species, which were inhibited by N-acetylcysteine (NAC). Hyperosmotic stress-induced NFkappaB activation as determined by IkappaBalpha degradation and NFkappaB DNA binding. NFkappaB activation and procaspase-3 and -9 fragmentation were prevented by NAC and IGF-1. However, this growth factor did not decrease ROS generation induced by hyperosmotic stress, suggesting that its actions over NFkappaB and caspase activation may be due to modulation of events downstream of ROS generation. We conclude that hyperosmotic stress induces ROS, which in turn activates NFkappaB and caspases. IGF-1 prevents NFkappaB activation by a ROS-independent mechanism.

Aldose reductase induced by hyperosmotic stress mediates cardiomyocyte apoptosis: differential effects of sorbitol and mannitol.

Cells adapt to hyperosmotic conditions by several mechanisms, including accumulation of sorbitol via induction of the polyol pathway. Failure to adapt to osmotic stress can result in apoptotic cell death. In the present study, we assessed the role of aldose reductase, the key enzyme of the polyol pathway, in cardiac myocyte apoptosis. Hyperosmotic stress, elicited by exposure of cultured rat cardiac myocytes to the nonpermeant solutes sorbitol and mannitol, caused identical cell shrinkage and adaptive hexose uptake stimulation. In contrast, only sorbitol induced the polyol pathway and triggered stress pathways as well as apoptosis-related signaling events. Sorbitol resulted in activation of the extracellular signal-regulated kinase (ERK), p54 c-Jun N-terminal kinase (JNK), and protein kinase B. Furthermore, sorbitol treatment resulting in induction and activation of aldose reductase, decreased expression of the antiapoptotic protein Bcl-xL, increased DNA fragmentation, and glutathione depletion. Apoptosis was attenuated by aldose reductase inhibition with zopolrestat and also by glutathione replenishment with N-acetylcysteine. In conclusion, our data show that hypertonic shrinkage of cardiac myocytes alone is not sufficient to induce cardiac myocyte apoptosis. Hyperosmolarity-induced cell death is sensitive to the nature of the osmolyte and requires induction of aldose reductase as well as a decrease in intracellular glutathione levels.

Mecanismos moleculares en la apoptosis del cardiomiocito y sus proyecciones patológicas y terapéuticas / Molecular mechanisms of cardiomyocyte apoptosis and their pathological and terapeutical perspectives

La apoptosis junto a la paraptosis y la necrosis constituyen las principales formas de muerte celular conocidas hasta la fecha. La apoptosis se caracteriza por una disminución del volumen celular y a laformación de cuerpos apoptóticos, manteniendo íntegra la membrana plasmática, evitando así el vaciamiento del contenido intracelular y el desarrollo de un proceso inflamatorio. En el cardiomiocito se han descrito dos vías apoptóticas: la tipo I (extrínseca o mediada a través de receptores de muerte) y la tipo II (intrínseca o mitocondrial). Ambas vías convergen en la caspasa 3, que es la responsable de la ejecución final de la apoptosis. Existe apoptosis en varias enfermedades cardíacas, como por ejemplo en la insuficiencia cardíaca de origen isquémico y no isquémico, en el infarto al miocardio y en las arritmias. Debido a que los cardiomiocitos son incapaces de proliferar, su muerte conduce a la pérdida de masa cardíaca, disminución de la capacidad contráctil del miocardio y remodelamiento. Dado que la apoptosis del cardiomiocito contribuye directamente a un deterioro funcional irreversible del corazón y favorece el desarrollo de diversas cardiopatías, el conocimiento de sus mecanismos y blancos moleculares proporcionará novedosas estrategias terapéuticas para la prevención y tratamiento de las diferentes cardiopatías