Clara Quiroga

Increased ER–mitochondrial coupling promotes mitochondrial respiration and bioenergetics during early phases of ER stress

Increasing evidence indicates that endoplasmic reticulum (ER) stress activates the adaptive unfolded protein response (UPR), but that beyond a certain degree of ER damage, this response triggers apoptotic pathways. The general mechanisms of the UPR and its apoptotic pathways are well characterized. However, the metabolic events that occur during the adaptive phase of ER stress, before the cell death response, remain unknown. Here, we show that, during the onset of ER stress, the reticular and mitochondrial networks are redistributed towards the perinuclear area and their points of connection are increased in a microtubule-dependent fashion. A localized increase in mitochondrial transmembrane potential is observed only in redistributed mitochondria, whereas mitochondria that remain in other subcellular zones display no significant changes. Spatial re-organization of these organelles correlates with an increase in ATP levels, oxygen consumption, reductive power and increased mitochondrial Ca2+ uptake. Accordingly, uncoupling of the organelles or blocking Ca2+ transfer impaired the metabolic response, rendering cells more vulnerable to ER stress. Overall, these data indicate that ER stress induces an early increase in mitochondrial metabolism that depends crucially upon organelle coupling and Ca2+ transfer, which, by enhancing cellular bioenergetics, establishes the metabolic basis for the adaptation to this response.

Glucose deprivation causes oxidative stress and stimulates aggresome formation and autophagy in cultured cardiac myocytes.

Aggresomes are dynamic structures formed when the ubiquitin-proteasome system is overwhelmed with aggregation-prone proteins. In this process, small protein aggregates are actively transported towards the microtubule-organizing center. A functional role for autophagy in the clearance of aggresomes has also been proposed. In the present work we investigated the molecular mechanisms involved on aggresome formation in cultured rat cardiac myocytes exposed to glucose deprivation. Confocal microscopy showed that small aggregates of polyubiquitinated proteins were formed in cells exposed to glucose deprivation for 6 h. However, at longer times (18 h), aggregates formed large perinuclear inclusions (aggresomes) which colocalized with gamma-tubulin (a microtubule-organizing center marker) and Hsp70. The microtubule disrupting agent vinblastine prevented the formation of these inclusions. Both small aggregates and aggresomes colocalized with autophagy markers such as GFP-LC3 and Rab24. Glucose deprivation stimulates reactive oxygen species (ROS) production and decreases intracellular glutathione levels. ROS inhibition by N-acetylcysteine or by the adenoviral overexpression of catalase or superoxide dismutase disrupted aggresome formation and autophagy induced by glucose deprivation. In conclusion, glucose deprivation induces oxidative stress which is associated with aggresome formation and activation of autophagy in cultured cardiac myocytes.

Cell Death and Survival Through the Endoplasmic Reticulum-Mitochondrial Axis

The endoplasmic reticulum has a central role in biosynthesis of a variety of proteins and lipids. Mitochondria generate ATP, synthesize and process numerous metabolites, and are key regulators of cell death. The architectures of endoplasmic reticulum and mitochondria change continually via the process of membrane fusion, fission, elongation, degradation, and renewal. These structural changes correlate with important changes in organellar function. Both organelles are capable of moving along the cytoskeleton, thus changing their cellular distribution. Numerous studies have demonstrated coordination and communication between mitochondria and endoplasmic reticulum. A focal point for these interactions is a zone of close contact between them known as the mitochondrial-associated endoplasmic reticulum membrane (MAM), which serves as a signaling juncture that facilitates calcium and lipid transfer between organelles. Here we review the emerging data on how communication between endoplasmic reticulum and mitochondria can modulate organelle function and determine cellular fate.

Mitochondria, Myocardial Remodeling, and Cardiovascular Disease

The process of muscle remodeling lies at the core of most cardiovascular diseases. Cardiac adaptation to pressure or volume overload is associated with a complex molecular change in cardiomyocytes which leads to anatomic remodeling of the heart muscle. Although adaptive at its beginnings, the sustained cardiac hypertrophic remodeling almost unavoidably ends in progressive muscle dysfunction, heart failure and ultimately death. One of the features of cardiac remodeling is a progressive impairment in mitochondrial function. The heart has the highest oxygen uptake in the human body and accordingly it has a large number of mitochondria, which form a complex network under constant remodeling in order to sustain the high metabolic rate of cardiac cells and serve as Ca2+ buffers acting together with the endoplasmic reticulum (ER). However, this high dependence on mitochondrial metabolism has its costs: when oxygen supply is threatened, high leak of electrons from the electron transport chain leads to oxidative stress and mitochondrial failure. These three aspects of mitochondrial function (Reactive oxygen species signaling, Ca2+ handling and mitochondrial dynamics) are critical for normal muscle homeostasis. In this article, we will review the latest evidence linking mitochondrial morphology and function with the process of myocardial remodeling and cardiovascular disease.

Dexamethasone-induced autophagy mediates muscle atrophy through mitochondrial clearance

Glucocorticoids, such as dexamethasone, enhance protein breakdown via ubiquitin–proteasome system. However, the role of autophagy in organelle and protein turnover in the glucocorticoid-dependent atrophy program remains unknown. Here, we show that dexamethasone stimulates an early activation of autophagy in L6 myotubes depending on protein kinase, AMPK, and glucocorticoid receptor activity. Dexamethasone increases expression of several autophagy genes, including ATG5, LC3, BECN1, and SQSTM1 and triggers AMPK-dependent mitochondrial fragmentation associated with increased DNM1L protein levels. This process is required for mitophagy induced by dexamethasone. Inhibition of mitochondrial fragmentation by Mdivi-1 results in disrupted dexamethasone-induced autophagy/mitophagy. Furthermore, Mdivi-1 increases the expression of genes associated with the atrophy program, suggesting that mitophagy may serve as part of the quality control process in dexamethasone-treated L6 myotubes. Collectively, these data suggest a novel role for dexamethasone-induced autophagy/mitophagy in the regulation of the muscle atrophy program.

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

We have recently shown that hyperosmotic stress activates p65/RelB NFκB in cultured cardiomyocytes with dichotomic actions on caspase activation and cell death. It remains unexplored how NFκB 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 NFκB and (b) if insulin‐like growth factor‐1 (IGF‐1) modulates ROS production and NFκB 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 NFκB activation as determined by IκBα degradation and NFκB DNA binding. NFκB 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 NFκB 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 NFκB and caspases. IGF‐1 prevents NFκB activation by a ROS‐independent mechanism.

Cardiomyocyte ryanodine receptor degradation by chaperone-mediated autophagy

AIMS Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins bearing the sequence KFERQ. These proteins are targeted by chaperones and delivered to lysosomes where they are translocated into the lysosomal lumen and degraded via the lysosome-associated membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy result in Danon disease characterized by hypertrophic cardiomyopathy. The ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte excitation-contraction and its dysfunction can lead to cardiac failure. Whether RyR2 is degraded by CMA is unknown. METHODS AND RESULTS To induce CMA, cultured neonatal rat cardiomyocytes were treated with geldanamycin (GA) to promote protein degradation through this pathway. GA increased LAMP-2A levels together with its redistribution and colocalization with Hsc70 in the perinuclear region, changes indicative of CMA activation. The inhibition of lysosomes but not proteasomes prevented the loss of RyR2. The recovery of RyR2 content after incubation with GA by siRNA targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis also revealed that the RyR2 sequence harbours six KFERQ motifs which are required for the recognition Hsc70 and its degradation via CMA. Our data suggest that presenilins are involved in RyR2 degradation by CMA. CONCLUSION These findings are consistent with a model in which oxidative damage of the RyR2 targets it for turnover by presenilins and CMA, which could lead to removal of damaged or leaky RyR2 channels.

Defective insulin signaling and mitochondrial dynamics in diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) is a common consequence of longstanding type 2 diabetes mellitus (T2DM) and encompasses structural, morphological, functional, and metabolic abnormalities in the heart. Myocardial energy metabolism depends on mitochondria, which must generate sufficient ATP to meet the high energy demands of the myocardium. Dysfunctional mitochondria are involved in the pathophysiology of diabetic heart disease. A large body of evidence implicates myocardial insulin resistance in the pathogenesis of DCM. Recent studies show that insulin signaling influences myocardial energy metabolism by impacting cardiomyocyte mitochondrial dynamics and function under physiological conditions. However, comprehensive understanding of molecular mechanisms linking insulin signaling and changes in the architecture of the mitochondrial network in diabetic cardiomyopathy is lacking. This review summarizes our current understanding of how defective insulin signaling impacts cardiac function in diabetic cardiomyopathy and discusses the potential role of mitochondrial dynamics.

Herp depletion protects from protein aggregation by up-regulating autophagy

Herp is an endoplasmic reticulum (ER) stress inducible protein that participates in the ER-associated protein degradation (ERAD) pathway. However, the contribution of Herp to other protein degradation pathways like autophagy and its connection to other types of stress responses remain unknown. Here we report that Herp regulates autophagy to clear poly-ubiquitin (poly-Ub) protein aggregates. Proteasome inhibition and glucose starvation (GS) led to a high level of poly-Ub protein aggregation that was drastically reduced by stably knocking down Herp (shHerp cells). The enhanced removal of poly-Ub inclusions protected cells from death caused by glucose starvation. Under basal conditions and increasingly after stress, higher LC3-II levels and GFP-LC3 puncta were observed in shHerp cells compared to control cells. Herp knockout cells displayed basal up-regulation of two essential autophagy regulators-Atg5 and Beclin-1, leading to increased autophagic flux. Beclin-1 up-regulation was due to a reduction in Hrd1 dependent proteasomal degradation, and not at transcriptional level. The consequent higher autophagic flux was necessary for the clearance of aggregates and for cell survival. We conclude that Herp operates as a relevant factor in the defense against glucose starvation by modulating autophagy levels. These data may have important implications due to the known up-regulation of Herp in pathological states such as brain and heart ischemia, both conditions associated to acute nutritional stress.

Hyperosmotic stress activates p65/RelB NFκB in cultured cardiomyocytes with dichotomic actions on caspase activation and cell death

NFκB is a participant in the process whereby cells adapt to stress. We have evaluated the activation of NFκB pathway by hyperosmotic stress in cultured cardiomyocytes and its role in the activation of caspase and cell death. Exposure of cultured rat cardiomyocytes to hyperosmotic conditions induced phosphorylation of IKKα/β as well as degradation of IκBα. All five members of the NFκB family were identified in cardiomyocytes. Analysis of the subcellular distribution of NFκB isoforms in response to hyperosmotic stress showed parallel migration of p65 and RelB from the cytosol to the nucleus. Measurement of the binding of NFκB to the consensus DNA κB‐site binding by EMSA revealed an oscillatory profile with maximum binding 1, 2 and 6 h after initiation of the hyperosmotic stress. Supershift analysis revealed that p65 and RelB (but not p50, p52 or cRel) were involved in the binding of NFκB to DNA. Hyperosmotic stress also resulted in activation of the NFκB‐lux reporter gene, transient activation of caspases 9 and 3 and phosphatidylserine externalization. The effect on cell viability was not prevented by ZVAD (a general caspase inhibitor). Blockade of NFκB with AdIκBα, an IκBα dominant negative overexpressing adenovirus, prevented activation of caspase 9 (more than that caspase 3) but did not affect cell death in hyperosmotically stressed cardiomyocytes. We conclude that hyperosmotic stress activates p65 and RelB NFκB isoforms and NFκB mediates caspase 9 activation in cardiomyocytes. However cell death triggered by hyperosmotic stress was caspase‐ and NFκB‐independent.