Ursodeoxycholic acid induces sarcopenia associated with decreased protein synthesis and autophagic flux.

BACKGROUND: Skeletal muscle generates force and movements and maintains posture. Under pathological conditions, muscle fibers suffer an imbalance in protein synthesis/degradation. This event causes muscle mass loss and decreased strength and muscle function, a syndrome known as sarcopenia. Recently, our laboratory described secondary sarcopenia in a chronic cholestatic liver disease (CCLD) mouse model. Interestingly, the administration of ursodeoxycholic acid (UDCA), a hydrophilic bile acid, is an effective therapy for cholestatic hepatic alterations. However, the effect of UDCA on skeletal muscle mass and functionality has never been evaluated, nor the possible involved mechanisms. METHODS: We assessed the ability of UDCA to generate sarcopenia in C57BL6 mice and develop a sarcopenic-like phenotype in C2C12 myotubes and isolated muscle fibers. In mice, we measured muscle strength by a grip strength test, muscle mass by bioimpedance and mass for specific muscles, and physical function by a treadmill test. We also detected the fiber's diameter and content of sarcomeric proteins. In C2C12 myotubes and/or isolated muscle fibers, we determined the diameter and troponin I level to validate the cellular effect. Moreover, to evaluate possible mechanisms, we detected puromycin incorporation, p70S6K, and 4EBP1 to evaluate protein synthesis and ULK1, LC3 I, and II protein levels to determine autophagic flux. The mitophagosome-like structures were detected by transmission electron microscopy. RESULTS: UDCA induced sarcopenia in healthy mice, evidenced by decreased strength, muscle mass, and physical function, with a decline in the fiber's diameter and the troponin I protein levels. In the C2C12 myotubes, we observed that UDCA caused a reduction in the diameter and content of MHC, troponin I, puromycin incorporation, and phosphorylated forms of p70S6K and 4EBP1. Further, we detected increased levels of phosphorylated ULK1, the LC3II/LC3I ratio, and the number of mitophagosome-like structures. These data suggest that UDCA induces a sarcopenic-like phenotype with decreased protein synthesis and autophagic flux. CONCLUSIONS: Our results indicate that UDCA induces sarcopenia in mice and sarcopenic-like features in C2C12 myotubes and/or isolated muscle fibers concomitantly with decreased protein synthesis and alterations in autophagic flux.

NEDD4-1 deficiency impairs satellite cell function during skeletal muscle regeneration.

BACKGROUND: Satellite cells are tissue-specific stem cells primarily responsible for the regenerative capacity of skeletal muscle. Satellite cell function and maintenance are regulated by extrinsic and intrinsic mechanisms, including the ubiquitin-proteasome system, which is key for maintaining protein homeostasis. In this context, it has been shown that ubiquitin-ligase NEDD4-1 targets the transcription factor PAX7 for proteasome-dependent degradation, promoting muscle differentiation in vitro. Nonetheless, whether NEDD4-1 is required for satellite cell function in regenerating muscle remains to be determined. RESULTS: Using conditional gene ablation, we show that NEDD4-1 loss, specifically in the satellite cell population, impairs muscle regeneration resulting in a significant reduction of whole-muscle size. At the cellular level, NEDD4-1-null muscle progenitors exhibit a significant decrease in the ability to proliferate and differentiate, contributing to the formation of myofibers with reduced diameter. CONCLUSIONS: These results indicate that NEDD4-1 expression is critical for proper muscle regeneration in vivo and suggest that it may control satellite cell function at multiple levels.

Disseminated intravascular coagulation phenotype is regulated by the TRPM7 channel during sepsis.

BACKGROUND: Sepsis is an uncontrolled inflammatory response against a systemic infection that results in elevated mortality, mainly induced by bacterial products known as endotoxins, producing endotoxemia. Disseminated intravascular coagulation (DIC) is frequently observed in septic patients and is associated with organ failure and death. Sepsis activates endothelial cells (ECs), promoting a prothrombotic phenotype contributing to DIC. Ion channel-mediated calcium permeability participates in coagulation. The transient reception potential melastatin 7 (TRPM7) non-selective divalent cation channel that also contains an α-kinase domain, which is permeable to divalent cations including Ca2+, regulates endotoxin-stimulated calcium permeability in ECs and is associated with increased mortality in septic patients. However, whether endothelial TRPM7 mediates endotoxemia-induced coagulation is not known. Therefore, our aim was to examine if TRPM7 mediates coagulation during endotoxemia. RESULTS: The results showed that TRPM7 regulated endotoxin-induced platelet and neutrophil adhesion to ECs, dependent on the TRPM7 ion channel activity and by the α-kinase function. Endotoxic animals showed that TRPM7 mediated neutrophil rolling on blood vessels and intravascular coagulation. TRPM7 mediated the increased expression of the adhesion proteins, von Willebrand factor (vWF), intercellular adhesion molecule 1 (ICAM-1), and P-selectin, which were also mediated by the TRPM7 α-kinase function. Notably, endotoxin-induced expression of vWF, ICAM-1 and P-selectin were required for endotoxin-induced platelet and neutrophil adhesion to ECs. Endotoxemic rats showed increased endothelial TRPM7 expression associated with a procoagulant phenotype, liver and kidney dysfunction, increased death events and an increased relative risk of death. Interestingly, circulating ECs (CECs) from septic shock patients (SSPs) showed increased TRPM7 expression associated with increased DIC scores and decreased survival times. Additionally, SSPs with a high expression of TRPM7 in CECs showed increased mortality and relative risk of death. Notably, CECs from SSPs showed significant results from the AUROC analyses for predicting mortality in SSPs that were better than the Acute Physiology and Chronic Health Evaluation II (APACHE II) and the Sequential Organ Failure Assessment (SOFA) scores. CONCLUSIONS: Our study demonstrates that sepsis-induced DIC is mediated by TRPM7 in ECs. TRPM7 ion channel activity and α-kinase function are required by DIC-mediated sepsis-induced organ dysfunction and its expression are associated with increased mortality during sepsis. TRPM7 appears as a new prognostic biomarker to predict mortality associated to DIC in SSPs, and as a novel target for drug development against DIC during infectious inflammatory diseases.

Cholic and deoxycholic acids induce mitochondrial dysfunction, impaired biogenesis and autophagic flux in skeletal muscle cells.

BACKGROUND: Skeletal muscle is sensitive to bile acids (BA) because it expresses the TGR5 receptor for BA. Cholic (CA) and deoxycholic (DCA) acids induce a sarcopenia-like phenotype through TGR5-dependent mechanisms. Besides, a mouse model of cholestasis-induced sarcopenia was characterised by increased levels of serum BA and muscle weakness, alterations that are dependent on TGR5 expression. Mitochondrial alterations, such as decreased mitochondrial potential and oxygen consumption rate (OCR), increased mitochondrial reactive oxygen species (mtROS) and unbalanced biogenesis and mitophagy, have not been studied in BA-induced sarcopenia. METHODS: We evaluated the effects of DCA and CA on mitochondrial alterations in C2C12 myotubes and a mouse model of cholestasis-induced sarcopenia. We measured mitochondrial mass by TOM20 levels and mitochondrial DNA; ultrastructural alterations by transmission electronic microscopy; mitochondrial biogenesis by PGC-1α plasmid reporter activity and protein levels by western blot analysis; mitophagy by the co-localisation of the MitoTracker and LysoTracker fluorescent probes; mitochondrial potential by detecting the TMRE probe signal; protein levels of OXPHOS complexes and LC3B by western blot analysis; OCR by Seahorse measures; and mtROS by MitoSOX probe signals. RESULTS: DCA and CA caused a reduction in mitochondrial mass and decreased mitochondrial biogenesis. Interestingly, DCA and CA increased LC3II/LC3I ratio and decreased autophagic flux concordant with raised mitophagosome-like structures. In addition, DCA and CA decreased mitochondrial potential and reduced protein levels in OXPHOS complexes I and II. The results also demonstrated that DCA and CA decreased basal, ATP-linked, FCCP-induced maximal respiration and spare OCR. DCA and CA also reduced the number of cristae. In addition, DCA and CA increased the mtROS. In mice with cholestasis-induced sarcopenia, TOM20, OXPHOS complexes I, II and III, and OCR were diminished. Interestingly, the OCR and OXPHOS complexes were correlated with muscle strength and bile acid levels. CONCLUSION: Our results showed that DCA and CA decreased mitochondrial mass, possibly by reducing mitochondrial biogenesis, which affects mitochondrial function, thereby altering potential OCR and mtROS generation. Some mitochondrial alterations were also observed in a mouse model of cholestasis-induced sarcopenia characterised by increased levels of BA, such as DCA and CA.

Upregulation of CCL5/RANTES Gene Expression in the Diaphragm of Mice with Cholestatic Liver Disease.

Chronic liver diseases are a group of pathologies affecting the liver with high prevalence worldwide. Among them, cholestatic chronic liver diseases (CCLD) are characterized by alterations in liver function and increased plasma bile acids. Secondary to liver disease, under cholestasis, is developed sarcopenia, a skeletal muscle dysfunction with decreased muscle mass, strength, and physical function. CCL5/RANTES is a chemokine involved in the immune and inflammatory response. Indeed, CCL5 is a myokine because it is produced by skeletal muscle. Several studies show that bile acids induce CCL5/RANTES expression in liver cells. However, it is unknown if the expression of CCL5/RANTES is changed in the skeletal muscle of mice with cholestatic liver disease. We used a murine model of cholestasis-induced sarcopenia by intake of hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC diet), in which we detected the mRNA levels for ccl5. We determined that mice fed the DDC diet presented high levels of serum bile acids and developed typical features of sarcopenia. Under these conditions, we detected the ccl5 gene expression in diaphragm muscle showing elevated mRNA levels compared to mice fed with a standard diet (chow diet). Our results collectively suggest an increased ccl5 gene expression in the diaphragm muscle concomitantly with elevated serum bile acids and the development of sarcopenia.

Physical Activity, Burnout, and Engagement in Latin American Students of Higher Education During the COVID-19 Pandemic.

The coronavirus-disease-2019 (COVID-19) pandemic has had a devastating physical and psychological impact on society, especially on students. In this study, we describe the levels of physical activity (Physical-Activity-Questionnaire-Short-Form (IPAQ-SF)), Burnout (School-Burnout-Inventory for students (SBI-U)) and engagement (Utrecht-Work-Engagement-Scale-9 items (UWES-9S)) in a cohort of Latin American higher education students during the COVID-19 pandemic in 2020. We also determined whether physical activity, Burnout, and engagement are related according to gender and area of study. Self-reported data from 571 Latin American students (64.79% women, 34.15% men; average age 25.24 ± 5.52 years) were collected via an online survey questionnaire. Spearman correlation analyses evaluated the associations between physical activity, Burnout, and engagement. Comparative analyses by gender and field of study were also performed. The results showed no correlation or association in the linear regression between the IPAQ-SF and SBI-U scores or between the IPAQ-SF and the UWES-9S scores. By gender, men had higher IPAQ-SF scores (p < 0.05) and reported higher intensity physical activity than women, but women had higher SBI-U scores (p < 0.05). No difference was found between men and women according to the UWES-9S scores (p = 0.28). There was also no difference in IPAQ-SF scores (p = 0.29) regarding the field of study. Our results suggest that women perform less physical activity than men, which is consistent with higher Burnout. However, physical activity was not associated with Burnout or engagement overall, which indicates that it was insufficient to prevent emotional stress in Latin American higher education students during a pandemic.

Impact of a Community-Based Pelvic Floor Kinesic Rehabilitation Program on the Quality of Life of Chilean Adult Women with Urinary Incontinence.

The pelvic floor forms the primary bottom tissue of the pelvic cavity. It comprises muscles that play a fundamental role in bowel and bladder emptying. Alterations of pelvic floor muscles will result in dysfunctions such as urinary incontinence (UI). Given the high prevalence of UI and its impact on the quality of life (QoL) in patients with pelvic floor muscle dysfunctions, it is necessary to implement public, community, and generalized programs focused on treating these dysfunctions. OBJECTIVE: To determine the effect of a community rehabilitation program on QoL, UI severity, and pelvic floor muscle strength in patients with UI. PATIENTS AND METHOD: A descriptive prospective cohort study. Twenty subjects between 44 and 75 years old with a diagnosis of UI, participants of a community kinesic rehabilitation program on the pelvic floor in Maipú, Santiago, Chile, were evaluated. These volunteers were intervened for six months, and QoL was measured with the 36-Item Short-Form Health Survey (SF-36) and International Consultation on Incontinence Questionnaire Short-Form (ICIQ-SF) scales, UI severity with the Sandvick test, and pelvic floor muscle strength with the Oxford scale. Patients were followed up three months post-intervention. RESULTS: Significant improvements were observed in all scales after applying for the community kinesic rehabilitation program, and the changes were maintained at a 3-month follow-up. CONCLUSIONS: Since the improvement in QoL, UI severity, and pelvic floor muscle strength after the intervention, it is relevant to consider the implementation of community programs aimed at education, screening, and early rehabilitation of these patients.

Bile Acids Alter the Autophagy and Mitogenesis in Skeletal Muscle Cells.

Muscle atrophy decreases muscle mass with the subsequent loss of muscle function. Among the mechanisms that trigger sarcopenia is mitochondrial dysfunction. Mitochondria, whose primary function is to produce ATP, are dynamic organelles that present the process of formation (mitogenesis) and elimination (mitophagy). Failure of any of these processes contributes to mitochondrial malfunction. Mitogenesis is mainly controlled by Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α), a transcriptional coactivator that regulates the expression of TFAM, which participates in mitogenesis. Mitophagy is a process of selective autophagy. Autophagy corresponds to a degradative pathway of protein complexes and organelles. Liver disease caused sarcopenia and increased bile acids in the blood. We demonstrated that the treatment with cholic (CA) or deoxycholic (DCA) bile acids generates mitochondrial dysfunction and loss of biomass. This work assessed whether CA and DCA alter autophagy and mitogenesis. For this, western blot evaluated the autophagy process by determining the protein levels of the LC3II/LC3I ratio. In addition, we assessed mitogenesis using a luciferase-coupled plasmid reporter for the PGC-1α promoter and the protein levels of TFAM by western blot. Our results indicate that treatment with CA or DCA induces autophagy, represented by an increase in the LC3II/LC3I ratio. In addition, a decreased autophagic flux was observed. On the other hand, when treated with CA or DCA, a decrease in the activity of the PGC-1α promoter was observed. However, the levels of TFAM increased in myotubes incubated with CA and DCA. Our results demonstrate that CA and DCA modulate autophagy ad mitogenesis in C2C12 myotubes.

Differential Fibrotic Response of Muscle Fibroblasts, Myoblasts, and Myotubes to Cholic and Deoxycholic Acids.

Fibrosis is a condition characterized by an increase in the components of the extracellular matrix (ECM). In skeletal muscle, the cells that participate in the synthesis of ECM are fibroblasts, myoblasts, and myotubes. These cells respond to soluble factors that increase ECM. Fibrosis is a phenomenon that develops in conditions of chronic inflammation, extensive lesions, or chronic diseases. A pathological condition with muscle weakness and increased bile acids (BA) in the blood is cholestatic chronic liver diseases (CCLD). Skeletal muscle expresses the membrane receptor for BA called TGR5. To date, muscle fibrosis in CCLD has not been evaluated. This study aims to assess whether BA can induce a fibrotic condition in muscle fibroblasts, myoblasts, and myotubes. The cells were incubated with deoxycholic (DCA) and cholic (CA) acids, and fibronectin protein levels were evaluated by Western blot. In muscle fibroblasts, both DCA and CA induced an increase in fibronectin protein levels. The same response was found in fibroblasts when activating TGR5 with the specific receptor agonist (INT-777). Interestingly, DCA reduced fibronectin protein levels in both myoblasts and myotubes, while CA did not show changes in fibronectin protein levels in myoblasts and myotubes. These results suggest that DCA and CA can induce a fibrotic phenotype in muscle-derived fibroblasts. On the other hand, DCA decreased the fibronectin in myoblasts and myotubes, whereas CA did not show any effect in these cell populations. Our results show that BA has different effects depending on the cell population to be analyzed.

Cholic acid and deoxycholic acid induce skeletal muscle atrophy through a mechanism dependent on TGR5 receptor.

Skeletal muscle atrophy is characterized by the degradation of myofibrillar proteins, such as myosin heavy chain or troponin. An increase in the expression of two muscle-specific E3 ligases, atrogin-1 and MuRF-1, and oxidative stress are involved in muscle atrophy. Patients with chronic liver diseases (CLD) develop muscle wasting. Several bile acids increase in plasma during cholestatic CLD, among them, cholic acid (CA) and deoxycholic acid (DCA). The receptor for bile acids, TGR5, is expressed in healthy skeletal muscles. TGR5 is involved in the regulation of muscle differentiation and metabolic changes. In this paper, we evaluated the participation of DCA and CA in the generation of an atrophic condition in myotubes and isolated fibers from the muscle extracted from wild-type (WT) and TGR5-deficient (TGR5-/- ) male mice. The results show that DCA and CA induce a decrease in diameter, and myosin heavy chain (MHC) protein levels, two typical atrophic features in C2 C12 myotubes. We also observed similar results when INT-777 agonists activated the TGR5 receptor. To evaluate the participation of TGR5 in muscle atrophy induced by DCA and CA, we used a culture of muscle fiber isolated from WT and TGR5-/- mice. Our results show that DCA and CA decrease the fiber diameter and MHC protein levels, and there is an increase in atrogin-1, MuRF-1, and oxidative stress in WT fibers. The absence of TGR5 in fibers abolished all these effects induced by DCA and CA. Thus, we demonstrated that CS and deoxycholic acid induce skeletal muscle atrophy through TGR5 receptor.

Correction: OxHDL controls LOX-1 expression and plasma membrane localization through a mechanism dependent on NOX/ROS/NF-κB pathway on endothelial cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

TRPM7 mediates kidney injury, endothelial hyperpermeability and mortality during endotoxemia.

Sepsis is the main cause of mortality in patients admitted to intensive care units. During sepsis, endothelial permeability is severely augmented, contributing to renal dysfunction and patient mortality. Ca2+ influx and the subsequent increase in intracellular [Ca2+]i in endothelial cells (ECs) are key steps in the establishment of endothelial hyperpermeability. Transient receptor potential melastatin 7 (TRPM7) ion channels are permeable to Ca2+ and are expressed in a broad range of cell types and tissues, including ECs and kidneys. However, the role of TRPM7 on endothelial hyperpermeability during sepsis has remained elusive. Therefore, we investigated the participation of TRPM7 in renal vascular hyperpermeability, renal dysfunction, and enhanced mortality induced by endotoxemia. Our results showed that endotoxin increases endothelial hyperpermeability and Ca2+ overload through the TLR4/NOX-2/ROS/NF-κB pathway. Moreover, endotoxin exposure was shown to downregulate the expression of VE-cadherin, compromising monolayer integrity and enhancing vascular hyperpermeability. Notably, endotoxin-induced endothelial hyperpermeability was substantially inhibited by pharmacological inhibition and specific suppression of TRPM7 expression. The endotoxin was shown to upregulate the expression of TRPM7 via the TLR4/NOX-2/ROS/NF-κB pathway and induce a TRPM7-dependent EC Ca2+ overload. Remarkably, in vivo experiments performed in endotoxemic animals showed that pharmacological inhibition and specific suppression of TRPM7 expression inhibits renal vascular hyperpermeability, prevents kidney dysfunction, and improves survival in endotoxemic animals. Therefore, our results showed that TRPM7 mediates endotoxemia-induced endothelial hyperpermeability, renal dysfunction, and enhanced mortality, revealing a novel molecular target for treating renal vascular hyperpermeability and kidney dysfunction during endotoxemia, sepsis, and other inflammatory diseases.

SARS-CoV-2/Renin-Angiotensin System: Deciphering the Clues for a Couple with Potentially Harmful Effects on Skeletal Muscle.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) has produced significant health emergencies worldwide, resulting in the declaration by the World Health Organization of the coronavirus disease 2019 (COVID-19) pandemic. Acute respiratory syndrome seems to be the most common manifestation of COVID-19. A high proportion of patients require intensive care unit admission and mechanical ventilation (MV) to survive. It has been well established that angiotensin-converting enzyme type 2 (ACE2) is the primary cellular receptor for SARS-CoV-2. ACE2 belongs to the renin-angiotensin system (RAS), composed of several peptides, such as angiotensin II (Ang II) and angiotensin (1-7) (Ang-(1-7)). Both peptides regulate muscle mass and function. It has been described that SARS-CoV-2 infection, by direct and indirect mechanisms, affects a broad range of organ systems. In the skeletal muscle, through unbalanced RAS activity, SARS-CoV-2 could induce severe consequences such as loss of muscle mass, strength, and physical function, which will delay and interfere with the recovery process of patients with COVID-19. This article discusses the relationship between RAS, SARS-CoV-2, skeletal muscle, and the potentially harmful consequences for skeletal muscle in patients currently infected with and recovering from COVID-19.

Protective Effect of Angiotensin 1-7 on Sarcopenia Induced by Chronic Liver Disease in Mice.

Sarcopenia associated with chronic liver disease (CLD) is one of the more common extrahepatic features in patients with these pathologies. Among the cellular alterations observed in the muscle tissue under CLD is the decline in the muscle strength and function, as well as the increased fatigue. Morphological changes, such as a decrease in the fiber diameter and transition in the fiber type, are also reported. At the molecular level, sarcopenia for CLD is characterized by: i) a decrease in the sarcomeric protein, such as myosin heavy chain (MHC); ii) an increase in the ubiquitin-proteasome system markers, such as atrogin-1/MAFbx1 and MuRF-1/TRIM63; iii) an increase in autophagy markers, such as LC3II/LC3I ratio. Among the regulators of muscle mass is the renin-angiotensin system (RAS). The non-classical axis of RAS includes the Angiotensin 1-7 [Ang-(1-7)] peptide and its receptor Mas, which in skeletal muscle has anti-atrophic effect in models of muscle wasting induced by immobilization, lipopolysaccharide, myostatin or angiotensin II. In this paper, we evaluated the effect of Ang-(1-7) on the sarcopenia by CLD in a murine model induced by the 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) hepatotoxin administered through diet. Our results show that Ang-(1-7) administration prevented the decline of the function and strength of muscle and increased the fatigue detected in the DDC-fed mice. Besides, we observed that the decreased fiber diameter and MHC levels, as well as the transition of fiber types, were all abolished by Ang-(1-7) in mice fed with DDC. Finally, Ang-(1-7) can decrease the atrogin-1 and MuRF-1 expression as well as the autophagy marker in mice treated with DDC. Together, our data support the protective role of Ang-(1-7) on the sarcopenia by CLD in mice.

Angiotensin (1-7) Decreases Myostatin-Induced NF-κB Signaling and Skeletal Muscle Atrophy.

Myostatin is a myokine that regulates muscle function and mass, producing muscle atrophy. Myostatin induces the degradation of myofibrillar proteins, such as myosin heavy chain or troponin. The main pathway that mediates protein degradation during muscle atrophy is the ubiquitin proteasome system, by increasing the expression of atrogin-1 and MuRF-1. In addition, myostatin activates the NF-κB signaling pathway. Renin-angiotensin system (RAS) also regulates muscle mass. Angiotensin (1-7) (Ang-(1-7)) has anti-atrophic properties in skeletal muscle. In this paper, we evaluated the effect of Ang-(1-7) on muscle atrophy and signaling induced by myostatin. The results show that Ang-(1-7) prevented the decrease of the myotube diameter and myofibrillar protein levels induced by myostatin. Ang-(1-7) also abolished the increase of myostatin-induced reactive oxygen species production, atrogin-1, MuRF-1, and TNF-α gene expressions and NF-κB signaling activation. Ang-(1-7) inhibited the activity mediated by myostatin through Mas receptor, as is demonstrated by the loss of all Ang-(1-7)-induced effects when the Mas receptor antagonist A779 was used. Our results show that the effects of Ang-(1-7) on the myostatin-dependent muscle atrophy and signaling are blocked by MK-2206, an inhibitor of Akt/PKB. Together, these data indicate that Ang-(1-7) inhibited muscle atrophy and signaling induced by myostatin through a mechanism dependent on Mas receptor and Akt/PKB.

Angiotensin-(1-7) Prevents Lipopolysaccharide-Induced Autophagy via the Mas Receptor in Skeletal Muscle.

Skeletal muscle atrophy, which occurs in lipopolysaccharide (LPS)-induced sepsis, causes a severe muscle function reduction. The increased autophagy contributes to sepsis-induced skeletal muscle atrophy in a model of LPS injection, increasing LC3II/LC3I ratio, autophagy flux, and autophagosomes. Angiotensin-(1-7) (Ang-(1-7)) has anti-atrophic effects via the Mas receptor in skeletal muscle. However, the impact of Ang-(1-7) on LPS-induced autophagy is unknown. In this study, we determined the effect of Ang-(1-7) on sepsis-induced muscle autophagy. C57BL6 wild-type (WT) mice and mice lacking the Mas receptor (KO Mas) were injected with LPS together with the systemic administration of Ang-(1-7) to determine autophagy in skeletal muscle. We also evaluated autophagy and p38 and c-Jun N-terminal kinase (JNK)activation. Our results show that Ang-(1-7) prevents LPS-induced autophagy in the diaphragm, tibialis anterior, and gastrocnemius of WT mice, which is demonstrated by a decrease in the LC3II/LC3I ratio and mRNA levels of lc3b and ctsl. This effect was lost in KO Mas mice, suggesting the role of the Mas receptor. The results in C2C12 cells show that Ang-(1-7) reduces several LPS-dependent effects, such as autophagy (LC3II/LC3I ratio, autophagic flux, and autophagosomes), activation of p38 and JNK, B-cell lymphoma-2 (BCL2) phosphorylation, and disassembly of the Beclin1/BCL2 complex. In conclusion, Ang-(1-7)/Mas receptor reduces LPS-induced autophagy in skeletal muscle. In vitro assays indicate that Ang-(1-7) prevents LPS-induced autophagy and modifies the MAPK signaling and the disassembly of a complex involved at the beginning of autophagy.

Sarcopenia Induced by Chronic Liver Disease in Mice Requires the Expression of the Bile Acids Membrane Receptor TGR5.

Sarcopenia is a condition of muscle dysfunction, commonly associated with chronic liver disease (CLD), characterized by a decline in muscle strength, the activation of the ubiquitin-proteasome system (UPS), and oxidative stress. We recently described a murine model of CLD-induced sarcopenia by intake of hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), which presents an increase in plasma bile acids (BA). BA induced skeletal muscle atrophy through a mechanism dependent on the Takeda G protein-coupled receptor 5 (TGR5) receptor. In the present study, we evaluated the role of TGR5 signaling in the development of sarcopenia using a model of DDC-induced CLD in C57BL6 wild-type (WT) mice and mice deficient in TGR5 expression (TGR5-/- mice). The results indicate that the decline in muscle function and contractibility induced by the DDC diet is dependent on TGR5 expression. TGR5 dependence was also observed for the decrease in fiber diameter and sarcomeric proteins, as well as for the fast-to-slow shift in muscle fiber type. UPS overactivation, indicated by increased atrogin-1/MAFbx (atrogin-1) and muscle RING-finger protein-1 (MuRF-1) protein levels and oxidative stress, was abolished in tibialis anterior muscles from TGR5-/- mice. Our results collectively suggest that all sarcopenia features induced by the DDC-supplemented diet in mice are dependent on TGR5 receptor expression.

OxHDL controls LOX-1 expression and plasma membrane localization through a mechanism dependent on NOX/ROS/NF-κB pathway on endothelial cells.

Systemic inflammatory diseases enhance circulating oxidative stress levels, which results in the oxidation of circulating high-density lipoprotein (oxHDL). Endothelial cell function can be negatively impacted by oxHDL, but the underlying mechanisms for this remain unclear. Some reports indicate that the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is also a receptor for oxHDL. However, it is unknown if oxHDL induces increased LOX-1 expression at the plasma membrane, as an event that supports endothelial dysfunction. Therefore, the aims of this study were to determine if oxHDL induces plasma-membrane level changes in LOX-1 and, if so, to describe the underlying mechanisms in endothelial cells. Our results demonstrate that the incubation of arterial or vein endothelial cells with oxHDL (and not HDL) induces the increase of LOX-1 expression at the plasma membrane; effect prevented by LOX-1 inhibition. Importantly, same results were observed in endothelial cells from oxHDL-treated rats. Furthermore, the observed oxHDL-induced LOX-1 expression is abolished by the down-regulation of NOX-2 expression with siRNA (and no others NOX isoforms), by the pharmacological inhibition of NAD(P)H oxidase (with DPI or apocynin) or by the inhibition of NF-κB transcription factor. Coherently, LOX-1 expression is augmented by the incubation of endothelial cells with H2O2 or GSSG even in absence of oxHDL, indicating that the NOX-2/ROS/ NF-κB axis is involved. Interestingly, oxHDL incubation also increases TNF-α expression, cytokine that induces LOX-1 expression. Thus, our results suggest a positive feedback mechanism for LOX-1 receptor during inflammatory condition where an oxidative burst will generate oxHDL from native HDL, activating LOX-1 receptor which in turn will increase the expression of NOX-2, TNF-α and LOX-1 receptor at the plasma membrane. In conclusion, oxHDL-induced translocation of LOX-1 to the plasma membrane could constitute an induction mechanism of endothelial dysfunction in systemic inflammatory diseases.

Endotoxemia-induced endothelial fibrosis inhibition improves hypotension, tachycardia, multiple organ dysfunction syndrome, cytokine response, oxidative stress, and survival.

Sepsis syndrome is the leading cause of mortality in critically ill patients admitted to intensive care. However, current therapies for sepsis treatment are unsatisfactory, and the mortality rate is still high. The main pathological characteristics observed during sepsis syndrome and endotoxemia include hypotension, tachycardia, multiple organ dysfunction syndrome (MODS), tissue damage, and cytokine and oxidative bursts. These conditions severely decrease the survival rates of endotoxemic patients. As a consequence of endotoxemia, large amounts of endotoxin circulate in the bloodstream throughout the vascular system and interact directly with endothelial cells that cover the inner wall of blood vessels. Endothelial cells exposed to lipopolysaccharides exhibit conversion to activated fibroblasts. By means of endotoxin-induced endothelial fibrosis, endothelial cells downregulate the expression of endothelial proteins and express fibrotic and ECM markers throughout endothelial protein expression reprogramming. Although endotoxin-induced endothelial fibrosis should, in theory, be detrimental to endothelial vascular function, the role of endothelial fibrosis in sepsis syndrome or endotoxemia is not known. Therefore, we employed a rat model to investigate whether the inhibition of endotoxin-induced endothelial fibrosis protects against endotoxemia and whether this inhibition increases survival. Our results show that the inhibition of endotoxin-induced endothelial fibrosis reduced both hypotension and tachycardia. Endotoxemia-induced MODS was also decreased when endothelial fibrosis was inhibited; treated rats showed normal kidney and liver function, inhibition of muscle mass wasting and normal glycemia. Liver and kidney histology was preserved, and organ fibrosis and fibrotic protein expression were reduced. Furthermore, pro-inflammatory cytokine secretion and NOX-2-mediated oxidative stress bursts were decreased when endothelial fibrosis was inhibited. Remarkably, the risk of death associated with sepsis syndrome at early and late time points was decreased when endotoxemia-induced endothelial fibrosis was inhibited, and a significant increase in survival was observed. These results reveal a potential novel treatment strategy to protect against sepsis syndrome and endotoxemia.

Circulating Endothelial Cells From Septic Shock Patients Convert to Fibroblasts Are Associated With the Resuscitation Fluid Dose and Are Biomarkers for Survival Prediction.

OBJECTIVES: To determine whether circulating endothelial cells from septic shock patients and from nonseptic shock patients are transformed in activated fibroblast by changing the expression level of endothelial and fibrotic proteins, whether the level of the protein expression change is associated with the amount of administered resuscitation fluid, and whether this circulating endothelial cell protein expression change is a biomarker to predict sepsis survival. DESIGN: Prospective study. SETTING: Medical-surgical ICUs in a tertiary care hospital. PATIENTS: Forty-three patients admitted in ICU and 22 healthy volunteers. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Circulating mature endothelial cells and circulating endothelial progenitor cells from septic shock and nonseptic shock patients showed evidence of endothelial fibrosis by changing the endothelial protein expression pattern. The endothelial proteins were downregulated, whereas fibroblast-specific markers were increased. The magnitude of the expression change in endothelial and fibrotic proteins was higher in the septic shock nonsurvivors patients but not in nonseptic shock. Interestingly, the decrease in the endothelial protein expression was correlated with the administered resuscitation fluid better than the Acute Physiology and Chronic Health Evaluation II and Sequential Organ Failure Assessment scores in the septic shock nonsurvivors patients but not in nonseptic shock. Notably, the significant difference between endothelial and fibrotic protein expression indicated a nonsurvival outcome in septic shock but not in nonseptic shock patients. Remarkably, area under the receiver operating characteristic curve analysis showed that endothelial protein expression levels predicted the survival outcome better than the Acute Physiology and Chronic Health Evaluation II and Sequential Organ Failure Assessment scores in septic shock but not in nonseptic shock patients. CONCLUSIONS: Circulating endothelial cells from septic shock patients are acutely converted into fibroblasts. Endothelial and fibrotic protein expression level are associated with resuscitation fluid administration magnitude and can be used as biomarkers for an early survival diagnosis of sepsis.