The STIM1 inhibitor ML9 disrupts basal autophagy in cardiomyocytes by decreasing lysosome content

Stromal-interaction molecule 1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) plays a key role in mediating cardiomyocyte hypertrophy, both in vitro and in vivo. Moreover, there is growing support for the contribution of SOCE to the Ca2+ overload associated with ischemia/reperfusion injury. Therefore, STIM1 inhibition is proposed as a novel target for controlling both hypertrophy and ischemia/reperfusion-induced Ca2+ overload. Our aim was to evaluate the effect of ML9, a STIM1 inhibitor, on cardiomyocyte viability. ML9 was found to induce cell death in cultured neonatal rat cardiomyocytes. Caspase-3 activation, apoptotic index and release of the necrosis marker lactate dehydrogenase to the extracellular medium were evaluated. ML9-induced cardiomyocyte death was not associated with increased intracellular ROS or decreased ATP levels. Moreover, treatment with ML9 significantly increased levels of the autophagy marker LC3-II, without altering Beclin1 or p62 protein levels. However, treatment with ML9 followed by bafilomycin-A1 did not produce further increases in LC3-II content. Furthermore, treatment with ML9 resulted in decreased LysoTracker (R) Green staining. Collectively, these data suggest that ML9-induced cardiomyocyte death is triggered by a ML9-dependent disruption of autophagic flux due to lysosomal dysfunction.FONDECYT, Chile 3150545 FONDAP 1513001

Therapeutic targeting of autophagy in myocardial infarction and heart failure

Introduction: Myocardial infarction (MI) is the leading cause of death. When MI is not lethal, heart failure (HF) is a major consequence with high prevalence and poor prognosis. The targeting of autophagy represents a potentially therapeutic approach for the treatment of both pathologies. Areas covered: PubMed searches were performed to discuss the current state of the art regarding the role of autophagy in MI and HF. We review available and potential approaches to modulate autophagy from a pharmacological and genetic perspective. We also discuss the targeting of autophagy in myocardial regeneration. Expert commentary: The targeting of autophagy has potential for the treatment of MI and HF. Autophagy is a process that takes place in virtually all cells of the body and thus, in order to evaluate this therapeutic approach in clinical trials, strategies that specifically target this process in the myocardium is required to avoid unwanted effects in other organs

ACE2 and vasoactive peptides: Novel players in cardiovascular/renal remodeling and hypertension

The renin–angiotensin system (RAS) is a key component of cardiovascular physiology and homeostasis due to its influence on the regulation of electrolyte balance, blood pressure, vascular tone and cardiovascular remodeling. Deregulation of this system contributes significantly to the pathophysiology of cardiovascular and renal diseases. Numerous studies have generated new perspectives about a noncanonical and protective RAS pathway that counteracts the proliferative and hypertensive effects of the classical angiotensin-converting enzyme (ACE)/angiotensin (Ang) II/angiotensin type 1 receptor (AT1R) axis. The key components of this pathway are ACE2 and its products, Ang-(1-7) and Ang-(1-9). These two vasoactive peptides act through the Mas receptor (MasR) and AT2R, respectively. The ACE2/Ang-(1-7)/MasR and ACE2/Ang-(1-9)/AT2R axes have opposite effects to those of the ACE/Ang II/AT1R axis, such as decreased proliferation and cardiovascular remodeling, increased production of nitric oxid

Transforming growth factor-beta and Forkhead box O transcription factors as cardiac fibroblast regulators

Fibroblasts play several homeostatic roles, including electrical coupling, paracrine signaling and tissue repair after injury. Fibroblasts have low secretory activity. However, in response to injury, they differentiate to myofibroblasts. These cells have an increased extracellular matrix synthesis and secretion, including collagen fibers, providing stiffness to the tissue. In pathological conditions myofibroblasts became resistant to apoptosis, remaining in the tissue, causing excessive extracellular matrix secretion and deposition, which contributes to the progressive tissue remodeling. Therefore, increased myofibroblast content within damaged tissue is a characteristic hallmark of heart, lung, kidney and liver fibrosis. Recently, it was described that cardiac fibroblast to myofibroblast differentiation is triggered by the transforming growth factor β1 (TGF-β1) through a Smad-independent activation of Forkhead box O (FoxO). FoxO proteins are a transcription factor family that includes FoxO1, FoxO3, FoxO4 and FoxO6. In several cells types, they play an important role in cell cycle arrest, oxidative stress resistance, cell survival, energy metabolism, and cell death. Here, we review the role of FoxO family members on the regulation of cardiac fibroblast proliferation and differentiation.Comision Nacional de Ciencia y Tecnologia (CONICYT), Chile, FONDECYT 1140329, FONDECYT 11160531, FONDECYT 113030

Legislative Documents

Also, variously referred to as: House bills; House documents; House legislative documents; legislative documents; General Court documents

Angiotensin II-regulated autophagy is required for vascular smooth muscle cell hypertrophy

Copyright © 2019 Mondaca-Ruff, Riquelme, Quiroga, Norambuena-Soto, Sanhueza-Olivares, Villar-Fincheira, Hernández-Díaz, Cancino-Arenas, San Martin, García, Lavandero and Chiong.Hypertension is a disease associated to increased plasma levels of angiotensin II (Ang II). Ang II can regulate proliferation, migration, ROS production and hypertrophy of vascular smooth muscle cells (VSMCs). However, the mechanisms by which Ang II can affect VSMCs remain to be fully elucidated. In this context, autophagy, a process involved in self-digestion of proteins and organelles, has been described to regulate vascular remodeling. Therefore, we sought to investigate if Ang II regulates VSMC hypertrophy through an autophagy-dependent mechanism. To test this, we stimulated A7r5 cell line and primary rat aortic smooth muscle cells with Ang II 100 nM and measured autophagic markers at 24 h by Western blot. Autophagosomes were quantified by visualizing fluorescently labeled LC3 using confocal microscopy. The results showed that treatment with Ang II increases Beclin-1, Vps34, Atg-12–Atg5, Atg4 and Atg7 protein levels, Beclin-1 phosphorylation, as well as the number of autophagic vesicles, suggesting that this peptide induces autophagy by activating phagophore initiation and elongation. These findings were confirmed by the assessment of autophagic flux by co-administering Ang II together with chloroquine (30 μM). Pharmacological antagonism of the angiotensin type 1 receptor (AT1R) with losartan and RhoA/Rho Kinase inhibition prevented Ang II-induced autophagy. Moreover, Ang II-induced A7r5 hypertrophy, evaluated by α-SMA expression and cell size, was prevented upon autophagy inhibition. Taking together, our results suggest that the induction of autophagy by an AT1R/RhoA/Rho Kinase-dependent mechanism contributes to Ang II-induced hypertrophy in VSMC

Association of N-cadherin levels and downstream effectors of Rho GTPases with dendritic spine loss induced by chronic stress in rat hippocampal neurons

© 2015 Wiley Periodicals, Inc. Chronic stress promotes cognitive impairment and dendritic spine loss in hippocampal neurons. In this animal model of depression, spine loss probably involves a weakening of the interaction between pre- and postsynaptic cell adhesion molecules, such as N-cadherin, followed by disruption of the cytoskeleton. N-cadherin, in concert with catenin, stabilizes the cytoskeleton through Rho-family GTPases. Via their effector LIM kinase (LIMK), RhoA and ras-related C3 botulinum toxin substrate 1 (RAC) GTPases phosphorylate and inhibit cofilin, an actin-depolymerizing molecule, favoring spine growth. Additionally, RhoA, through Rho kinase (ROCK), inactivates myosin phosphatase through phosphorylation of the myosin-binding subunit (MYPT1), producing actomyosin contraction and probable spine loss. Some micro-RNAs negatively control the translation of specific mRNAs involved in Rho GTPase signaling. For example, miR-138 indirectly activates RhoA, and miR-134 reduces

Autophagy mediates TNF-α-induced secretion of IL-6 in A7r5 cells.

<p>(<b>A</b>) Determination of IL-6 mRNA using RT-qPCR in A7r5 cells treated with TNF-α (100 ng/mL) for 30 min, 1 and 6 h (n = 6; *<i>p</i><0.05, **<i>p</i><0.01 vs 0 h). (<b>B</b>) Determination of IL-6 using ELISA assay in A7r5 cells treated with TNF-α (100 ng/mL) for 24 and 48 h (n = 4; **<i>p</i><0.01, ***<i>p</i><0.001 vs 0 h) or (<b>C</b>) co-administered with or without chloroquine (CQ, 20 μmol/L) during the last 4 h of the 24 h stimulus with TNF-α (n = 4; ***<i>p</i><0.001 vs control). Data are expressed as mean ± SEM and analyzed by one-way ANOVA, followed by Dunnett and Tukey post-tests.</p

TNF-α induces dedifferentiation of A7r5 cells by an autophagy-dependent pathway.

<p>(<b>A</b>) Western blot analysis of α-SMA and SM22 (n = 3–4; *<i>p</i><0.05, **<i>p</i><0.01 vs control) or (<b>B</b>) collagen type I and osteopontin (n = 3–4; **p<0.01 vs control) in A7r5 cells incubated with TNF-α (100 ng/mL) for 48 h in the presence or absence of siScramble and siBeclin1. GAPDH was used as loading control. (<b>C</b>) Visualization of actin filaments in A7r5 cells stained with rhodamine-phalloidin after treatment with TNF-α (100 ng/mL) for 48 h in the presence or absence of chloroquine (CQ, 5 μmol/L) during the last 24 h of TNF-α stimulus. Lower panel represent a fluorescence intensity profile of the lines depicted on the images. Data are expressed as mean ± SEM and analyzed by two-way ANOVA, followed by Holm Sidak post-test.</p

TNF-α requires autophagy to induce migration in A7r5 cells.

<p>(<b>A</b>) Assessment of migration by the wound healing and transwell assays in A7r5 cells stimulated with TNF-α (100 ng/mL) for 24 h in the presence or absence of chloroquine (CQ, 20 μmol/L) during the last 4 h of TNF-α stimulus (n = 4; **<i>p</i><0.01 vs control) or (<b>C</b>) siScramble and siBeclin1 for 24 h (n = 4; **<i>p</i><0.01 vs control). Migration was visualized using a phase contrast microscope (upper panels of <b>A</b> and <b>C</b>). The results of the wound healing and transwell assays were quantified by measuring wound width and the number of cells that migrated through the Boyden chamber, respectively (lower panels of <b>A</b> and <b>C</b>). (<b>B</b>) Zymography analysis of matrix metalloproteinase 9 (MMP-9) in A7r5 cells stimulated with TNF-α (100 ng/mL) for 24 h (n = 3; *<i>p</i><0.05 vs control). Data are expressed as mean ± SEM and analyzed by one-way ANOVA, followed by Holm Sidak (<b>A</b> and <b>C</b>) and Dunnett (<b>B</b>) post-tests.</p