Juana Martínez-Llorens

Inflammatory cells and apoptosis in respiratory and limb muscles of patients with COPD

Discrepancies exist regarding the involvement of cellular inflammation and apoptosis in the muscle dysfunction of chronic obstructive pulmonary disease (COPD) patients with preserved body composition. We explored whether levels of inflammatory cells and apoptosis were increased in both respiratory and limb muscles of COPD patients without nutritional abnormalities. In the vastus lateralis, external intercostals, and diaphragms of severe and moderate COPD patients with normal body composition, and in healthy subjects, intramuscular leukocytes and macrophage levels were determined (immunohistochemistry). Muscle structure was also evaluated. In the diaphragm and vastus lateralis of severe and moderate COPD patients and controls, apoptotic nuclei were explored using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, electron microscopy, and caspase-3 expression. In COPD patients compared with controls, diaphragm and intercostal levels of inflammatory cells were extremely low and not significantly different. However, in the vastus lateralis of the severe patients, inflammatory cell counts, although also very low, were significantly greater. In those patients, TUNEL-positive nuclei levels were also significantly greater in diaphragms and vastus lateralis. A significant inverse relationship was found between quadriceps TUNEL-positive nuclei levels and muscle force. Ultrastructural apoptotic nuclei revealed no differences in respiratory or limb muscles between COPD patients and controls. Muscle caspase-3 expression did not differ between patients and controls. In severe COPD patients with preserved body composition, while increased apoptotic nuclei seems to be a contributor to their muscle dysfunction, cellular inflammation does not. The increased numbers of TUNEL-positive nuclei in their muscles suggest that they may also be exposed to a continuous repair/remodeling process.

Muscle dysfunction and exercise limitation in adolescent idiopathic scoliosis

Adolescent idiopathic scoliosis (AIS) can lead to ventilatory restriction, respiratory muscle weakness and exercise limitation. However, both the causes and the extent of muscle dysfunction remain unclear. The aim of our study is to describe muscle weakness and its relationship to lung function and tolerance to exercise in AIS patients. Lung and muscle function, together with exercise capacity, were assessed in 60 patients with pronounced spinal deformity (>40°) and in 25 healthy volunteers. Patients with AIS had only mild to moderate abnormal ventilatory patterns, the most frequent of which were restrictive abnormalities. The function of respiratory and limb muscles and exercise capacity were below normal limits in AIS patients, and were significantly lower than in controls. Exercise capacity was found to correlate with the function of inspiratory, expiratory, upper limb and lower limb muscles which, in addition, were reciprocally interrelated. Multivariate analysis showed that lower limb muscle function is the main contributor to exercise intolerance. There appeared to be no connection between spinal deformity and lung function, muscle function or exercise capacity. We conclude that AIS patients show generalised muscle dysfunction which contributes to the reduction in their exercise capacity, even in the absence of severe ventilatory impairment.

Quadriceps muscle weakness and atrophy are associated with a differential epigenetic profile in advanced COPD

Epigenetic mechanisms regulate muscle mass and function in models of muscle dysfunction and atrophy. We assessed whether quadriceps muscle weakness and atrophy are associated with a differential expression profile of epigenetic events in patients with advanced COPD (chronic obstructive pulmonary disease). In vastus lateralis (VL) of sedentary severe COPD patients (n=41), who were further subdivided into those with (n=25) and without (n=16) muscle weakness and healthy controls (n=19), expression of muscle-enriched miRNAs, histone acetyltransferases (HATs) and deacetylases (HDACs), growth and atrophy signalling markers, total protein and histone acetylation, transcription factors, small ubiquitin-related modifier (SUMO) ligases and muscle structure were explored. All subjects were clinically evaluated. Compared with controls, in VL of all COPD together and in muscle-weakness patients, expression of miR-1, miR-206 and miR-27a, levels of lysine-acetylated proteins and histones and acetylated histone 3 were increased, whereas expression of HDAC3, HDAC4, sirtuin-1 (SIRT-1), IGF-1 (insulin-like growth factor-1) were decreased, Akt (v-akt murine thymoma viral oncogene homologue 1) expression did not differ, follistatin expression was greater, whereas myostatin expression was lower, serum reponse factor (SRF) expression was increased and fibre size of fast-twitch fibres was significantly reduced. In VL of severe COPD patients with muscle weakness and atrophy, epigenetic events regulate muscle differentiation rather than proliferation and muscle growth and atrophy signalling, probably as feedback mechanisms to prevent those muscles from undergoing further atrophy. Lysine-hyperacetylation of histones may drive enhanced protein catabolism in those muscles. These findings may help design novel therapeutic strategies (enhancers of miRNAs promoting myogenesis and acetylation inhibitors) to selectively target muscle weakness and atrophy in severe COPD.

Do epigenetic events take place in the vastus lateralis of patients with mild chronic obstructive pulmonary disease

Muscle dysfunction is a major comorbidity in Chronic Obstructive Pulmonary Disease (COPD). Several biological mechanisms including epigenetic events regulate muscle mass and function in models of muscle atrophy. Investigations conducted so far have focused on the elucidation of biological mechanisms involved in muscle dysfunction in advanced COPD. We assessed whether the epigenetic profile may be altered in the vastus lateralis of patients with mild COPD, normal body composition, and mildly impaired muscle function and exercise capacity. In vastus lateralis (VL) of mild COPD patients with well-preserved body composition and in healthy age-matched controls, expression of DNA methylation, muscle-enriched microRNAs, histone acetyltransferases (HTAs) and deacetylases (HDACs), protein acetylation, small ubiquitin-related modifier (SUMO) ligases, and muscle structure were explored. All subjects were clinically evaluated. Compared to healthy controls, in the VL of mild COPD patients, muscle function and exercise capacity were moderately reduced, DNA methylation levels did not differ, miR-1 expression levels were increased and positively correlated with both forced expiratory volume in one second (FEV1) and quadriceps force, HDAC4 protein levels were increased, and muscle fiber types and sizes were not different. Moderate skeletal muscle dysfunction is a relevant feature in patients with mild COPD and preserved body composition. Several epigenetic events are differentially expressed in the limb muscles of these patients, probably as an attempt to counterbalance the underlying mechanisms that alter muscle function and mass. The study of patients at early stages of their disease is of interest as they are a target for timely therapeutic interventions that may slow down the course of the disease and prevent the deleterious effects of major comorbidities.

Epigenetic Mechanisms in Respiratory Muscle Dysfunction of Patients with Chronic Obstructive Pulmonary Disease

Epigenetic events are differentially expressed in the lungs and airways of patients with chronic obstructive pulmonary disease (COPD). Moreover, epigenetic mechanisms are involved in the skeletal (peripheral) muscle dysfunction of COPD patients. Whether epigenetic events may also regulate respiratory muscle dysfunction in COPD remains unknown. We hypothesized that epigenetic mechanisms would be differentially expressed in the main inspiratory muscle (diaphragm) of patients with COPD of a wide range of disease severity compared to healthy controls. In diaphragm muscle specimens (thoracotomy due to lung localized neoplasms) of sedentary patients with mild-to-moderate and severe COPD, with preserved body composition, and sedentary healthy controls, expression of muscle-enriched microRNAs, histone acetyltransferases (HATs) and deacetylases (HDACs), total DNA methylation and protein acetylation, small ubiquitin-related modifier (SUMO) ligases, muscle-specific transcription factors, and muscle structure were explored. All subjects were also clinically evaluated: lung and muscle functions and exercise capacity. Compared to healthy controls, patients exhibited moderate airflow limitation and diffusion capacity, and reduced exercise tolerance and transdiaphragmatic strength. Moreover, in the diaphragm of the COPD patients, muscle-specific microRNA expression was downregulated, while HDAC4 and myocyte enhancer factor (MEF)2C protein levels were higher, and DNA methylation levels, muscle fiber types and sizes did not differ between patients and controls. In the main respiratory muscle of COPD patients with a wide range of disease severity and normal body composition, muscle-specific microRNAs were downregulated, while HDAC4 and MEF2C levels were upregulated. It is likely that these epigenetic events act as biological adaptive mechanisms to better overcome the continuous inspiratory loads of the respiratory system in COPD. These findings may offer novel therapeutic strategies to specifically target respiratory muscle dysfunction in patients with COPD.

Vastus Lateralis Fiber Shift Is an Independent Predictor of Mortality in Chronic Obstructive Pulmonary Disease

To the Editor: Quadriceps weakness and atrophy is present in approximately 30% of patients with chronic obstructive pulmonary disease (COPD) in secondary care (1, 2). The quadriceps also displays a shift in fiber type so that there are fewer type I (oxidative) fibers and more type II (glycolytic) fibers (3). Pulmonary rehabilitation only partially addresses this fiber shift (4). Muscle mass (5) and strength (6) are both associated with increased mortality, but the prognostic significance of fiber shift is unknown. In a retrospective multicenter analysis of 392 patients from four sites (see Tables E1–E4 in the online supplement), mortality data were collated, as part of audit procedures, on outpatients with stable COPD who had undergone a vastus lateralis biopsy between 1995 and 2013. Data from these subjects have been previously published (e.g., References 2, 4, 5). Fiber proportion, reported as the percentage of type II fibers (type II fiber %), was established by immunohistochemistry. Fiber shift, evaluated as a dichotomous variable, was considered to have occurred when the proportion of type II fibers was greater than 68% (men) or greater than 65% (women) based on normal ranges established from an age-matched healthy population published by Natanek and colleagues (3). Body mass index (BMI), fat-free mass index (FFMI), dominant leg isometric quadriceps maximum voluntary contraction (QMVC and QMVC/BMI), mid-thigh cross-sectional area determined by computed tomography scan (MTCSA), residual volume normalized to total lung capacity (RV/TLC), and percent predicted value for the carbon monoxide transfer factor corrected for hemoglobin (TLCOc), when available, were included in subanalyses. Data were analyzed for the whole dataset and also after splitting the group into those with an FEV1 less than 50% predicted and those with an FEV1 greater than or equal to 50% predicted. Further details on the methodology and statistical analyses are presented in the online supplement. Some of the results of this study have been previously reported in abstract form (7). Patients were followed up for a median of 1,699 days (127–6,601 d); 102 of 392 (26.7%) patients died during follow-up (Table E6). Cohort characteristics are presented in Tables 1 and ​and22 and Tables E1–E5. One hundred fifty-one patients had Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage I/II disease and 241 had GOLD stage III/IV disease. Those who died were older and had a lower FEV1 % predicted, and there was a greater male preponderance (Table E6A). One hundred seventy-seven (45.1%) of the patients had fiber shift. The patients who died had a higher percentage of type II fibers (69.5% [62.2, 76.3%] vs. 66.0% [54.0, 74.2%]; P = 0.002) and a higher proportion of them exhibited fiber shift (58% vs. 41%, P = 0.004). BMI, FFMI, QMVC, MTCSA, and TLCOc were all lower, and RV/TLC higher, in those who died (Table E6B). Table 1. Core Characteristics of the Cohort (n = 392) in Addition to Univariate and Multivariate Analyses Including Type II Fiber Proportion Dichotomized into the Occurrence of Fiber Shift Table 2. Core Characteristics of the Cohort (n = 392) in Addition to Univariate and Multivariate Analyses Including Type II Fiber Proportion as a Continuous Measure In the cohort considered as a whole, both type II fiber % and the presence of fiber shift were univariate predictors of mortality, as were age and FEV1 % predicted (Tables 1 and ​and2).2). In a multivariate analysis including fiber shift as a dichotomous variable, fiber shift was retained, as were age and FEV1 % predicted, Table 1. When age, FEV1 % predicted, and type II fiber % were entered into a multivariate analysis, age and FEV1 % predicted were retained as independent predictors, but the association between fiber type and mortality just missed statistical significance (Table 2). The relationship between FEV1 and fiber proportion is shown in Figure 1A, and survival as a function of fiber shift, adjusted for age and FEV1, is shown in Figure 1B. Additional data regarding other lung function and muscle parameters are presented in Tables E7–E10. FEV1 expressed in liters and TLCOc were also univariate predictors of mortality; however, RV/TLC was not. When including TLCOc in the analysis (n = 209), fiber shift, age, FEV1 % predicted, and TLCOc were all independent predictors of mortality. In other subanalyses, BMI, FFMI, QMVC, QMVC/BMI, and MTCSA were not univariate predictors of mortality. Figure 1. (A) The relationship between type II fiber percentage and FEV1 % predicted (dashed lines demonstrate the 95% confidence interval), and (B) survival curves for those with fiber shift (n = 177) and those without fiber shift (n = 215) after adjusting for ... When limiting the analysis to those with an FEV1 greater than or equal to 50% predicted, age was the only predictor of mortality (hazard ratio [HR], 1.16; 95% CI, 1.07, 1.25; P < 0.0001; Table E11). In a multivariate analysis confined to those with an FEV1 less than 50%, fiber shift was retained as an independent predictor (HR, 1.71; 95% CI, 1.08, 2.71; P = 0.02), as were age (HR, 1.06; 95% CI, 1.03, 1.09; P < 0.0001), and FEV1 % predicted (HR, 0.96; 95% CI, 0.94, 0.99; P = 0.002; Table E12A). In a separate analysis confined to those with an FEV1 less than 50%, type II fiber % was not retained as an independent predictor (HR, 1.014; 95% CI, 0.996, 1.032; P = 0.13), whereas age and FEV1 % predicted were (Table E12B). Fiber shift in the vastus lateralis of patients with COPD was associated with increased mortality, although this association was weaker when lung function and age were included in the analysis. This finding was pronounced in patients with GOLD stage III/IV disease but undetectable in those with GOLD stage I/II disease. The relationship between skeletal muscle atrophy (5) and weakness (6) with mortality has been previously noted in COPD. However, we believe the present analysis is timely because we (3) and others (8) have recently shown that the nature of skeletal muscle involvement in COPD is heterogeneous rather than uniform. No prior study has related quadriceps biopsy appearances to long-term outcome in COPD. Given the known relationship between exercise capacity and survival (9), these data are consistent with our prior studies, which demonstrated a relationship between fiber shift (although not fiber atrophy) and impaired exercise capacity (3) and functional performance (10). Nevertheless, it remains unclear whether fiber shift causes poor exercise tolerance or is a manifestation of exercise intolerance and reduced physical activity, which are both associated with increased mortality in COPD (9, 11). Both concepts can be supported by in vivo models that demonstrate that muscle disuse results in type I to type II fiber shift (12) and that fiber shift toward a type I fiber predominance increases exercise performance (13). Due to the retrospective nature of the current analysis, exercise performance and physical activity data were not available for inclusion in this report, so a causative role for fiber shift in mortality cannot be demonstrated from this study. A prospective study would have been preferable and could also have considered other factors of relevance, including pulmonary rehabilitation over the intervening period. Despite the limitations of the current study, it is doubtful that a prospective study of comparable size and duration will ever be done. Interest in pharmacological management of skeletal muscle dysfunction is growing (14), and addressing fiber shift may eventually become a therapeutic possibility. Further studies to address whether the reversal of fiber shift is of benefit are of value.

Injury of peripheral muscles in smokers with chronic obstructive pulmonary disease.

Muscle injury has clinical relevance in diseased individuals because it is associated with muscle dysfunction in terms of decreased strength and/or endurance. This study was aimed at answering three questions: whether the presence of chronic obstructive pulmonary disease (COPD) is associated with peripheral muscle injury; whether muscle injury is associated with some of the relevant functional impairment in the muscles; and whether muscle injury can be solely justified by deconditioning. Twenty-one male COPD patients were eligible for the study. Seven healthy volunteers recruited from the general population were included as controls. Function of the quadriceps muscle was assessed through specific single-leg exercise (strength and endurance). Cellular (light microscopy) and subcellular (electron microscopy) techniques were used to evaluate muscle injury on biopsies from the vastus lateralis muscle. Signs of injury were found in muscles from both control and COPD patients, not only in cases showing severe airflow obstruction but also in the mild or moderate stages of the disease. Current smoking and presence of COPD were significantly associated with increased injury of the muscle as assessed by light and electron microscopy techniques. The authors conclude that peripheral muscle injury is evident in mild, moderate, and severe stages of COPD even in the absence of respiratory failure, hypercapnia, chronic steroid treatment, low body weight, or some coexisting disease. These findings support the theory that systemic factors with deleterious effect are acting on peripheral muscles of smokers with COPD, increasing the susceptibility of the muscle fibers to membrane and sarcomere injury.

Short-Stay Respiratory Unit: A New Option for Inpatient Care

OBJECTIVE The current pressure on conventional inpatient care units represents a very serious problem for respiratory medicine departments. The aim of this study was to analyze the impact of a new instrument --a short-stay respiratory unit-- on the quality of care delivered by the respiratory medicine department of a tertiary care hospital. MATERIAL AND METHODS The short-stay respiratory unit consisted of 4 conventional hospital beds managed by a pulmonologist. The beds were only used to treat patients diagnosed with exacerbation of chronic obstructive pulmonary disease or bronchial asthma, community-acquired pneumonia, or suspected lung cancer, and stays were intended to only last for up less than to 4 days. Analyzing a range of healthcare quality variables, we compared the quality of care delivered during the first 6 months the unit was in operation (October 2005 to March 2006) to that delivered in the same period 12 months earlier. RESULTS The study included 147 patients admitted to the short-stay unit. The mean (SD) age of the patients was 64 (17) years and 79% were men. The mean length of stay was 3.3 (1.6) days--only 1.4% of patients stayed for longer than 4 days--and the readmission rate was 2.7%. There were no deaths. The existence of the short-stay respiratory unit led to a 30% decrease in the overall mean length of stay in the respiratory medicine department (11.8 [4.6] vs 8.3 [2.6] days; P< .001), and the readmission rate fell from 21% to 15% (P< .05). While there were no differences in mortality between the 2 periods, the complexity of diseases treated by the respiratory medicine department showed an increase of 9.2% (P< .001). CONCLUSIONS A short-stay respiratory unit can improve the efficiency of care delivered by a respiratory medicine department by reducing both the mean duration of hospitalization and the readmission rate, without a reduction in the complexity of diseases treated or a need for additional resources.

Activation of Satellite Cells in the Intercostal Muscles of Patients With Chronic Obstructive Pulmonary Disease

OBJECTIVE The respiratory muscles of patients with chronic obstructive pulmonary disease (COPD) display evidence of structural damage in parallel with signs of adaptation. We hypothesized that this can only be explained by the simultaneous activation of satellite cells. The aim of this study was to analyze the number and activation of those cells along with the expression of markers of microstructural damage that are frequently associated with regeneration. PATIENTS AND METHODS The study included 8 patients with severe COPD (mean [SD] forced expiratory volume in 1 second, 33% [9%] of predicted) and 7 control subjects in whom biopsies were performed of the external intercostal muscle. The samples were analyzed by light microscopy to assess muscle fiber phenotype, electron microscopy to identify satellite cells, and real-time polymerase chain reaction to analyze the expression of the following markers: insulin-like growth factor 1, mechano growth factor, and embryonic and perinatal myosin heavy chains (MHC) as markers of microstructural damage; Pax-7 and m-cadherin as markers of the presence and activation of satellite cells, respectively; and MHC-I, IIa, and IIx as determinants of muscle fiber phenotype. RESULTS The patients had larger fibers than healthy subjects (54 [6] vs 42 [4] microm(2); P< .01) with a similar or slightly increased proportion of satellite cells, as measured by ultrastructural analysis (4.3% [1%] vs 3.7% [3.5%]; P>.05) or expression of Pax-7 (5.5 [4.1] vs 1.6 [0.8] arbitrary units [AU]; P< .05). In addition, there was greater activation of satellite cells in the patients, as indicated by increased expression of m-cadherin (3.8 [2.1] vs 1.0 [1.2] AU; P=.05). This was associated with increased expression of markers of microstructural damage: insulin-like growth factor 1, 0.35 (0.34) vs 0.09 (0.08) AU (P< .05); mechano growth factor, 0.45 (0.55) vs 0.13 (0.17) AU (P=.05). CONCLUSIONS The intercostal muscles of patients with severe COPD show indirect signs of microstructural damage accompanied by satellite cell activation. This suggests the presence of ongoing cycles of lesion and repair that could partially explain the maintenance of the structural properties of the muscle.

Interpretation of the approximate entropy using fixed tolerance values as a measure of amplitude variations in biomedical signals

A new method for the quantification of amplitude variations in biomedical signals through moving approximate entropy is presented. Unlike the usual method to calculate the approximate entropy (ApEn), in which the tolerance value (r) varies based on the standard deviation of each moving window, in this work ApEn has been computed using a fixed value of r. We called this method, moving approximate entropy with fixed tolerance values: ApEnf. The obtained results indicate that ApEnf allows determining amplitude variations in biomedical data series. These amplitude variations are better determined when intermediate values of tolerance are used. The study performed in diaphragmatic mechanomyographic signals shows that the ApEnf curve is more correlated with the respiratory effort than the standard RMS amplitude parameter. Furthermore, it has been observed that the ApEnf parameter is less affected by the existence of impulsive, sinusoidal, constant and Gaussian noises in comparison with the RMS amplitude parameter.