Sergio Lavandero

Regulation of autophagy by the inositol trisphosphate receptor.

The reduction of intracellular 1,4,5-inositol trisphosphate (IP3) levels stimulates autophagy, whereas the enhancement of IP3 levels inhibits autophagy induced by nutrient depletion. Here, we show that knockdown of the IP3 receptor (IP3R) with small interfering RNAs and pharmacological IP3R blockade is a strong stimulus for the induction of autophagy. The IP3R is known to reside in the membranes of the endoplasmic reticulum (ER) as well as within ER–mitochondrial contact sites, and IP3R blockade triggered the autophagy of both ER and mitochondria, as exactly observed in starvation-induced autophagy. ER stressors such as tunicamycin and thapsigargin also induced autophagy of ER and, to less extent, of mitochondria. Autophagy triggered by starvation or IP3R blockade was inhibited by Bcl-2 and Bcl-XL specifically targeted to ER but not Bcl-2 or Bcl-XL proteins targeted to mitochondria. In contrast, ER stress-induced autophagy was not inhibited by Bcl-2 and Bcl-XL. Autophagy promoted by IP3R inhibition could not be attributed to a modulation of steady-state Ca2+ levels in the ER or in the cytosol, yet involved the obligate contribution of Beclin-1, autophagy-related gene (Atg)5, Atg10, Atg12 and hVps34. Altogether, these results strongly suggest that IP3R exerts a major role in the physiological control of autophagy.

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.

The IKK complex contributes to the induction of autophagy

In response to stress, cells start transcriptional and transcription‐independent programs that can lead to adaptation or death. Here, we show that multiple inducers of autophagy, including nutrient depletion, trigger the activation of the IKK (IκB kinase) complex that is best known for its essential role in the activation of the transcription factor NF‐κB by stress. Constitutively active IKK subunits stimulated autophagy and transduced multiple signals that operate in starvation‐induced autophagy, including the phosphorylation of AMPK and JNK1. Genetic inhibition of the nuclear translocation of NF‐κB or ablation of the p65/RelA NF‐κB subunit failed to suppress IKK‐induced autophagy, indicating that IKK can promote the autophagic pathway in an NF‐κB‐independent manner. In murine and human cells, knockout and/or knockdown of IKK subunits (but not that of p65) prevented the induction of autophagy in response to multiple stimuli. Moreover, the knockout of IKK‐β suppressed the activation of autophagy by food deprivation or rapamycin injections in vivo, in mice. Altogether, these results indicate that IKK has a cardinal role in the stimulation of autophagy by physiological and pharmacological stimuli.

The inositol-1,4,5-trisphosphate receptor regulates autophagy through its interaction with Beclin 1

The inositol 1,4,5-trisphosphate receptor (IP3R) is a major regulator of apoptotic signaling. Through interactions with members of the Bcl-2 family of proteins, it drives calcium (Ca2+) transients from the endoplasmic reticulum (ER) to mitochondria, thereby establishing a functional and physical link between these organelles. Importantly, the IP3R also regulates autophagy, and in particular, its inhibition/depletion strongly induces macroautophagy. Here, we show that the IP3R antagonist xestospongin B induces autophagy by disrupting a molecular complex formed by the IP3R and Beclin 1, an interaction that is increased or inhibited by overexpression or knockdown of Bcl-2, respectively. An effect of Beclin 1 on Ca2+ homeostasis was discarded as siRNA-mediated knockdown of Beclin 1 did not affect cytosolic or luminal ER Ca2+ levels. Xestospongin B- or starvation-induced autophagy was inhibited by overexpression of the IP3R ligand-binding domain, which coimmunoprecipitated with Beclin 1. These results identify IP3R as a new regulator of the Beclin 1 complex that may bridge signals converging on the ER and initial phagophore formation.

Insulin-like Growth Factor-I Rapidly Activates Multiple Signal Transduction Pathways in Cultured Rat Cardiac Myocytes

In response to insulin-like growth factor-I (IGF-I), neonatal rat cardiac myocytes exhibit a hypertrophic response. The elucidation of the IGF-I signal transduction system in these cells remains unknown. We show here that cardiac myocytes present a single class of high affinity receptors (12,446 ± 3,669 binding sites/cell) with a dissociation constant of 0.36 ± 0.10 nm. Two different β-subunits of IGF-I receptor were detected, and their autophosphorylation was followed by increases in the phosphotyrosine content of extracellular signal-regulated kinases (ERKs), insulin receptor substrate 1, phospholipase C-γ1, and phosphatidylinositol 3-kinase. IGF-I transiently activates c-Raf in cultured neonatal cardiac myocytes, whereas A-raf is activated much less than c-Raf. Two peaks of ERK activity (ERK1 and ERK2) were resolved in cardiac myocytes treated with IGF-I by fast protein liquid chromatography, both being stimulated by IGF-I (with EC50values for the stimulation of ERK1 and ERK2 by IGF-I of 0.10 and 0.12 nm, respectively). Maximal activation of ERK2 (12-fold) and ERK1 (8.3-fold) activities was attained after a 5-min exposure to IGF-I. Maximal activation of p90 S6 kinase by IGF-I was achieved after 10 min, and then the activity decreased slowly. Interestingly, IGF-I stimulates incorporation of [3H]phenylalanine (1.6-fold) without any effect on [3H]thymidine incorporation. These data suggest that IGF-I activates multiple signal transduction pathways in cardiac myocytes some of which may be relevant to the hypertrophic response of the heart.

Cardiomyocyte death: mechanisms and translational implications

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide. Although treatments have improved, development of novel therapies for patients with CVD remains a major research goal. Apoptosis, necrosis, and autophagy occur in cardiac myocytes, and both gradual and acute cell death are hallmarks of cardiac pathology, including heart failure, myocardial infarction, and ischemia/reperfusion. Pharmacological and genetic inhibition of autophagy, apoptosis, or necrosis diminishes infarct size and improves cardiac function in these disorders. Here, we review recent progress in the fields of autophagy, apoptosis, and necrosis. In addition, we highlight the involvement of these mechanisms in cardiac pathology and discuss potential translational implications.

Senescence, apoptosis or autophagy? When a damaged cell must decide its path--a mini-review.

Many features of aging result from the incapacity of cells to adapt to stress conditions. When damage accumulates irreversibly, mitotic cells from renewable tissues rely on either of two mechanisms to avoid replication. They can permanently arrest the cell cycle (cellular senescence) or trigger cell death programs. Apoptosis (self-killing) is the best-described form of programmed cell death, but autophagy (self-eating), which is a lysosomal degradation pathway essential for homeostasis, reportedly contributes to cell death as well. Unlike mitotic cells, postmitotic cells like neurons or cardiomyocytes cannot become senescent since they are already terminally differentiated. The fate of these cells entirely depends on their ability to cope with stress. Autophagy then operates as a major homeostatic mechanism to eliminate damaged organelles, long-lived or aberrant proteins and superfluous portions of the cytoplasm. In this mini-review, we briefly summarize the molecular networks that allow damaged cells either to adapt to stress or to engage in programmed-cell-death pathways.

Metabolic stress–induced activation of FoxO1 triggers diabetic cardiomyopathy in mice

The leading cause of death in diabetic patients is cardiovascular disease; diabetic cardiomyopathy is typified by alterations in cardiac morphology and function, independent of hypertension or coronary disease. However, the molecular mechanism that links diabetes to cardiomyopathy is incompletely understood. Insulin resistance is a hallmark feature of diabetes, and the FoxO family of transcription factors, which regulate cell size, viability, and metabolism, are established targets of insulin and growth factor signaling. Here, we set out to evaluate a possible role of FoxO proteins in diabetic cardiomyopathy. We found that FoxO proteins were persistently activated in cardiac tissue in mice with diabetes induced either genetically or by high-fat diet (HFD). FoxO activity was critically linked with development of cardiomyopathy: cardiomyocyte-specific deletion of FoxO1 rescued HFD-induced declines in cardiac function and preserved cardiomyocyte insulin responsiveness. FoxO1-depleted cells displayed a shift in their metabolic substrate usage, from free fatty acids to glucose, associated with decreased accumulation of lipids in the heart. Furthermore, we found that FoxO1-dependent downregulation of IRS1 resulted in blunted Akt signaling and insulin resistance. Together, these data suggest that activation of FoxO1 is an important mediator of diabetic cardiomyopathy and is a promising therapeutic target for the disease.

Endoplasmic reticulum and the unfolded protein response: dynamics and metabolic integration.

The endoplasmic reticulum (ER) is a dynamic intracellular organelle with multiple functions essential for cellular homeostasis, development, and stress responsiveness. In response to cellular stress, a well-established signaling cascade, the unfolded protein response (UPR), is activated. This intricate mechanism is an important means of re-establishing cellular homeostasis and alleviating the inciting stress. Now, emerging evidence has demonstrated that the UPR influences cellular metabolism through diverse mechanisms, including calcium and lipid transfer, raising the prospect of involvement of these processes in the pathogenesis of disease, including neurodegeneration, cancer, diabetes mellitus and cardiovascular disease. Here, we review the distinct functions of the ER and UPR from a metabolic point of view, highlighting their association with prevalent pathologies.

Histone Deacetylase Inhibition Blunts Ischemia/Reperfusion Injury by Inducing Cardiomyocyte Autophagy

Background— Reperfusion accounts for a substantial fraction of the myocardial injury occurring with ischemic heart disease. Yet, no standard therapies are available targeting reperfusion injury. Here, we tested the hypothesis that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor approved for cancer treatment by the US Food and Drug Administration, will blunt reperfusion injury. Methods and Results— Twenty-one rabbits were randomly assigned to 3 groups: (1) vehicle control, (2) SAHA pretreatment (1 day before and at surgery), and (3) SAHA treatment at the time of reperfusion only. Each arm was subjected to ischemia/reperfusion surgery (30 minutes coronary ligation, 24 hours reperfusion). In addition, cultured neonatal and adult rat ventricular cardiomyocytes were subjected to simulated ischemia/reperfusion to probe mechanism. SAHA reduced infarct size and partially rescued systolic function when administered either before surgery (pretreatment) or solely at the time of reperfusion. SAHA plasma concentrations were similar to those achieved in patients with cancer. In the infarct border zone, SAHA increased autophagic flux, assayed in both rabbit myocardium and in mice harboring an RFP-GFP-LC3 transgene. In cultured myocytes subjected to simulated ischemia/reperfusion, SAHA pretreatment reduced cell death by 40%. This reduction in cell death correlated with increased autophagic activity in SAHA-treated cells. RNAi-mediated knockdown of ATG7 and ATG5, essential autophagy proteins, abolished SAHA’s cardioprotective effects. Conclusions— The US Food and Drug Administration–approved anticancer histone deacetylase inhibitor, SAHA, reduces myocardial infarct size in a large animal model, even when delivered in the clinically relevant context of reperfusion. The cardioprotective effects of SAHA during ischemia/reperfusion occur, at least in part, through the induction of autophagic flux.