Martín Angulo

High CO2 Levels Cause Skeletal Muscle Atrophy via AMP-activated Kinase (AMPK), FoxO3a Protein, and Muscle-specific Ring Finger Protein 1 (MuRF1)

Background: CO2 retention and skeletal muscle atrophy occur in patients with lung diseases and are associated with poor clinical outcomes. Results: Hypercapnia leads to AMPK/FoxO3a/MuRF1-dependent muscle fiber size reduction. Conclusion: Hypercapnia activates a signaling pathway leading to skeletal muscle atrophy. Significance: High CO2 levels directly activate a proteolytic program of skeletal muscle atrophy which is of relevance to patients with lung diseases. Patients with chronic obstructive pulmonary disease, acute lung injury, and critical care illness may develop hypercapnia. Many of these patients often have muscle dysfunction which increases morbidity and impairs their quality of life. Here, we investigated whether hypercapnia leads to skeletal muscle atrophy. Mice exposed to high CO2 had decreased skeletal muscle wet weight, fiber diameter, and strength. Cultured myotubes exposed to high CO2 had reduced fiber diameter, protein/DNA ratios, and anabolic capacity. High CO2 induced the expression of MuRF1 in vivo and in vitro, whereas MuRF1−/− mice exposed to high CO2 did not develop muscle atrophy. AMP-activated kinase (AMPK), a metabolic sensor, was activated in myotubes exposed to high CO2, and loss-of-function studies showed that the AMPKα2 isoform is necessary for muscle-specific ring finger protein 1 (MuRF1) up-regulation and myofiber size reduction. High CO2 induced AMPKα2 activation, triggering the phosphorylation and nuclear translocation of FoxO3a, and leading to an increase in MuRF1 expression and myotube atrophy. Accordingly, we provide evidence that high CO2 activates skeletal muscle atrophy via AMPKα2-FoxO3a-MuRF1, which is of biological and potentially clinical significance in patients with lung diseases and hypercapnia.

HOIL-1L functions as the PKCζ ubiquitin ligase to promote lung tumor growth

RATIONALE Protein kinase C zeta (PKCζ) has been reported to act as a tumor suppressor. Deletion of PKCζ in experimental cancer models has been shown to increase tumor growth. However, the mechanisms of PKCζ down-regulation in cancerous cells have not been previously described. OBJECTIVES To determine the molecular mechanisms that lead to decreased PKCζ expression and thus increased survival in cancer cells and tumor growth. METHODS The levels of expression of heme-oxidized IRP2 ubiquitin ligase 1L (HOIL-1L), HOIL-1-interacting protein (HOIP), Shank-associated RH domain-interacting protein (SHARPIN), and PKCζ were analyzed by Western blot and/or quantitative real-time polymerase chain reaction in different cell lines. Coimmunoprecipitation experiments were used to demonstrate the interaction between HOIL-1L and PKCζ. Ubiquitination was measured in an in vitro ubiquitination assay and by Western blot with specific antibodies. The role of hypoxia-inducible factor (HIF) was determined by gain/loss-of-function experiments. The effect of HOIL-1L expression on cell death was investigated using RNA interference approaches in vitro and on tumor growth in mice models. Increased HOIL-1L and decreased PKCζ expression was assessed in lung adenocarcinoma and glioblastoma multiforme and documented in several other cancer types by oncogenomic analysis. MEASUREMENTS AND MAIN RESULTS Hypoxia is a hallmark of rapidly growing solid tumors. We found that during hypoxia, PKCζ is ubiquitinated and degraded via the ubiquitin ligase HOIL-1L, a component of the linear ubiquitin chain assembly complex (LUBAC). In vitro ubiquitination assays indicate that HOIL-1L ubiquitinates PKCζ at Lys-48, targeting it for proteasomal degradation. In a xenograft tumor model and lung cancer model, we found that silencing of HOIL-1L increased the abundance of PKCζ and decreased the size of tumors, suggesting that lower levels of HOIL-1L promote survival. Indeed, mRNA transcript levels of HOIL-1L were elevated in tumor of patients with lung adenocarcinoma, and in a lung adenocarcinoma tissue microarray the levels of HOIL-1L were associated with high-grade tumors. Moreover, we found that HOIL-1L expression was regulated by HIFs. Interestingly, the actions of HOIL-1L were independent of LUBAC. CONCLUSIONS These data provide first evidence of a mechanism of cancer cell adaptation to hypoxia where HIFs regulate HOIL-1L, which targets PKCζ for degradation to promote tumor survival. We provided a proof of concept that silencing of HOIL-1L impairs lung tumor growth and that HOIL-1L expression predicts survival rate in cancer patients suggesting that HOIL-1L is an attractive target for cancer therapy.

Interrater Reliability and Diagnostic Performance of Subjective Evaluation of Sublingual Microcirculation Images by Physicians and Nurses: A Multicenter Observational Study.

ABSTRACT Introduction: This was a cross-sectional multicenter study to investigate the ability of physicians and nurses from three different countries to subjectively evaluate sublingual microcirculation images and thereby discriminate normal from abnormal sublingual microcirculation based on flow and density abnormalities. Methods: Forty-five physicians and 61 nurses (mean age, 36 ± 10 years; 44 males) from three different centers in The Netherlands (n = 61), Uruguay (n = 12), and Japan (n = 33) were asked to subjectively evaluate a sample of 15 microcirculation videos randomly selected from an experimental model of endotoxic shock in pigs. All videos were first analyzed offline using the A.V.A. software by an independent, experienced investigator and were categorized as good, bad, or very bad microcirculation based on the microvascular flow index, perfused capillary density, and proportion of perfused capillaries. Then, the videos were randomly assigned to the examiners, who were instructed to subjectively categorize each image as good, bad, or very bad. An interrater analysis was performed, and sensitivity and specificity tests were calculated to evaluate the proportion of A.V.A. score abnormalities that the examiners correctly identified. Results: The &kgr; statistics indicated moderate agreement in the evaluation of microcirculation abnormalities using three categories, i.e., good, bad, or very bad (&kgr; = 0.48), and substantial agreement using two categories, i.e., normal (good) and abnormal (bad or very bad) (&kgr; = 0.66). There was no significant difference between the &kgr; three and &kgr; two statistics. We found that the examiner’s subjective evaluations had good diagnostic performance and were highly sensitive (84%; 95% confidence interval, 81%–86%) and specific (87%; 95% confidence interval, 84%–90%) for sublingual microcirculatory abnormalities as assessed using the A.V.A. software. Conclusions: The subjective evaluations of sublingual microcirculation by physicians and nurses agreed well with a conventional offline analysis and were highly sensitive and specific for sublingual microcirculatory abnormalities.

Adenosine triphosphate–dependent calcium signaling during ventilator-induced lung injury is amplified by hypercapnia

ABSTRACT Adenosine triphosphate (ATP) is released by alveolar epithelial cells during ventilator-induced lung injury (VILI) and regulates fluid transport across epithelia. High CO2 levels are observed in patients with “permissive hypercapnia,” which inhibits alveolar fluid reabsorption (AFR) in alveolar epithelial cells. The authors set out to determine whether VILI affects AFR and whether the purinergic pathway is modulated in cells exposed to hypercapnia. Control group was compared against VILI (tidal volume [Vt] = 35 mL/kg, zero positive end-expiratory pressure [PEEP]) and protective ventilation (Vt = 6 mL/kg, PEEP = 10 cm H2O) groups. Lung mechanics, histology, and AFR were evaluated. Alveolar epithelial cells (AECs) were loaded with Fura 2-AM to measure intracellular calcium in the presence ATP (10 μM) at 5% or 10% CO2 as compared with baseline. High tidal volume ventilation impairs lung mechanics and AFR. Hypercapnia (HC) increases intracellular calcium levels in response to ATP stimulation. HC + ATP is the most detrimental combination decreasing AFR. Purinergic signaling in AECs is modulated by high CO2 levels via increased cytosolic calcium. The authors reason that this modulation may play a role in the impairment of alveolar epithelial functions induced by hypercapnia.

El salbutamol mejora la fuerza diafragmática en la sepsis experimental

Objetivo: La sepsis grave se acompa?a en un alto porcentaje de casos de insuficiencia respiratoria aguda, donde la debilidad de los m?sculos respiratorios desempe?a un papel importante. Los m?sculos respiratorios debilitados y sometidos a una carga mec?nica aumentada pueden evolucionar a fatiga muscular con agravamiento de la insuficiencia respiratoria. Los f?rmacos adren?rgicos ?2, al mejorar la fuerza de contracci?n muscular, podr?an ser de utilidad en la prevenci?n y el manejo de la insuficiencia respiratoria de pacientes con sepsis. El objetivo de este trabajo ha sido estudiar los efectos del salbutamol en la funci?n diafragm?tica en un modelo animal de sepsis peritoneal. Material y m?todos: Se estudiaron 3 grupos de animales: a) grupo control (n = 7), al que se realiz? laparotom?a mediana sin abordaje visceral; b) grupo sepsis (n = 10), al que se indujo sepsis peritoneal por ligadura y punci?n cecal (LPC), y c) grupo salbutamol (n = 7), en el que la sepsis peritoneal se trat? con salbutamol (LPC + salbutamol). Los par?metros hemodin?micos y los gases sangu?neos se midieron in vivo. La funci?n diafragm?tica se evalu? in vitro. Resultados: El salbutamol aument? el flujo a?rtico y la frecuencia card?aca a la vez que disminuy? la presi?n arterial media en la sepsis peritoneal (p < 0,05). La sepsis determin? una ca?da significativa de la fuerza diafragm?tica tanto antes como despu?s de un protocolo de fatiga muscular. El tratamiento con salbutamol mejor? la fuerza de contracci?n muscular en ambos casos (p < 0,05). Conclusiones: El uso de agentes adren?rgicos ?2 como el salbutamol mejora la funci?n diafragm?tica durante la sepsis experimental. Los mecanismos de esta mejor?a deben estudiarse en mayor profundidad.

Salbutamol Improves Diaphragm Force Generation in Experimental Sepsis

OBJECTIVE In a high percentage of cases, severe sepsis is accompanied by acute respiratory failure, in which weakness of the respiratory muscles plays an important role. Weakened respiratory muscles that are subjected to an increased mechanical load may develop muscle fatigue, with exacerbation of the respiratory failure. Because beta2-adrenergic drugs increase muscle contraction force, they may play a role in preventing and managing respiratory failure in septic patients. Our aim was to study the effects of salbutamol on diaphragm function in an animal model of peritoneal sepsis. MATERIAL AND METHODS The study included 3 groups of animals: a) a control group (n=7), in which the animals underwent a median laparotomy without visceral manipulation; b) a septic group (n=10), in which peritoneal sepsis was induced by cecal ligation and puncture (CLP); and c) a salbutamol group (n=7), in which peritoneal sepsis (CLP) was treated with salbutamol. Hemodynamic parameters and blood gases were measured in vivo. Diaphragm function was evaluated in vitro. RESULTS Salbutamol increased aortic blood flow and heart rate while it reduced mean arterial pressure in the animals with peritoneal sepsis (P< .05). Sepsis produced a significant drop in diaphragmatic force both before and after the application of a muscle-fatigue protocol. Treatment with salbutamol improved muscle contraction force before and after application of the protocol (P< .05). CONCLUSIONS The use of beta2-adrenergic drugs such as salbutamol improves diaphragm function in experimental sepsis. The mechanisms that produce this improvement require further study.

El salbutamol mejora la contractilidad diafragmática en la obstrucción crónica de la vía aérea

INTRODUCTION Chronic airflow obstruction in conditions such as chronic obstructive pulmonary disease is associated with respiratory muscle dysfunction. Our aim was to study the effects of salbutamol-a beta-adrenergic agonist known to improve muscle strength in physiologic and pathologic conditions-on diaphragm contractility in an animal model of chronic airway obstruction achieved by tracheal banding. MATERIALS AND METHODS Twenty-four Sprague-Dawley rats were randomized into a control group and 3 tracheal banding groups, 1 that received acute salbutamol treatment, 1 that received chronic salbutamol treatment, and 1 that received nothing. Arterial blood gases, acid-base balance, and in vitro diaphragmatic contractility were evaluated by measuring peak twitch tension, contraction time, contraction velocity, half-relaxation time, relaxation velocity, and force-frequency curves. RESULTS The 3 study groups had significantly reduced arterial pH and increased PaCO2 and bicarbonate levels compared to the control group (P<.05). The untreated tracheal banding group had significantly reduced peak twitch tension and contraction velocity, and a significantly lower force-frequency curve in comparison with the other groups (P<.05). The chronic treatment group had a higher relaxation velocity than the untreated study group (P<.05). The mean (SE) peak twitch tension values were 6.46 (0.90)N/cm(2) for the control group, 3.28 (0.55)N/cm(2) for the untreated tracheal banding group, 6.18 (0.71)N/cm(2) for the acute treatment group, and 7.09 (0.59)N/cm(2) for the chronic treatment group. CONCLUSIONS Diaphragmatic dysfunction associated with chronic airflow obstruction improves with both the acute and chronic administration of salbutamol. The mechanisms involved in respiratory muscle dysfunction warrant further study.

Salbutamol Improves Diaphragmatic Contractility in Chronic Airway Obstruction

Introduction: Chronic airfl ow obstruction in conditions such as chronic obstructive pulmonary disease is associated with respiratory muscle dysfunction. Our aim was to study the effects of salbutamol—a β2adrenergic agonist known to improve muscle strength in physiologic and pathologic conditions—on diaphragm contractility in an animal model of chronic airway obstruction achieved by tracheal banding. Materials and Methods: Twenty-four Sprague-Dawley rats were randomized into a control group and 3 tracheal banding groups, 1 that received acute salbutamol treatment, 1 that received chronic salbutamol treatment, and 1 that received nothing. Arterial blood gases, acid-base balance, and in vitro diaphragmatic contractility were evaluated by measuring peak twitch tension, contraction time, contraction velocity, halfrelaxation time, relaxation velocity, and force-frequency curves. Results: The 3 study groups had signifi cantly reduced arterial pH and increased PaCO2 and bicarbonate levels compared to the control group (P<.05). The untreated tracheal banding group had signifi cantly reduced peak twitch tension and contraction velocity, and a signifi cantly lower force-frequency curve in comparison with the other groups (P<.05). The chronic treatment group had a higher relaxation velocity than the untreated study group (P<.05). The mean (SE) peak twitch tension values were 6.46 (0.90) N/cm 2

Hypercapnia increases airway smooth muscle contractility via caspase-7–mediated miR-133a–RhoA signaling

Elevated carbon dioxide (hypercapnia) activates complex signaling pathways in airway smooth muscle cells, resulting in airway constriction. Giving lungs a breath of fresh air Severe lung disorders such as chronic obstructive pulmonary disease (COPD) are often associated with hypoventilation that can lead to hypercapnia, an elevation of carbon dioxide (CO2) in the bloodstream. Although hypercapnia has been associated with increased mortality among COPD patients, whether the increased CO2 plays a role in disease pathophysiology is unclear. Now, Shigemura and colleagues show that CO2 may act as a signaling molecule in lungs and that hypercapnia promoted smooth muscle cell contractility in mice. In COPD patients, hypercapnia was associated with increased airway resistance that was ameliorated by ventilation. The results suggest that reducing hypercapnia with noninvasive ventilation might have therapeutic effects in lung disorders. The elevation of carbon dioxide (CO2) in tissues and the bloodstream (hypercapnia) occurs in patients with severe lung diseases, including chronic obstructive pulmonary disease (COPD). Whereas hypercapnia has been recognized as a marker of COPD severity, a role for hypercapnia in disease pathogenesis remains unclear. We provide evidence that CO2 acts as a signaling molecule in mouse and human airway smooth muscle cells. High CO2 activated calcium-calpain signaling and consequent smooth muscle cell contraction in mouse airway smooth muscle cells. The signaling was mediated by caspase-7–induced down-regulation of the microRNA-133a (miR-133a) and consequent up-regulation of Ras homolog family member A and myosin light-chain phosphorylation. Exposure of wild-type, but not caspase-7–null, mice to hypercapnia increased airway contraction and resistance. Deletion of the Caspase-7 gene prevented hypercapnia-induced airway contractility, which was restored by lentiviral transfection of a miR-133a antagonist. In a cohort of patients with severe COPD, hypercapnic patients had higher airway resistance, which improved after correction of hypercapnia. Our data suggest a specific molecular mechanism by which the development of hypercapnia may drive COPD pathogenesis and progression.