Study design & participants
This study is a cross-sectional analysis of the baseline data collected from the selected participants with or without COPD at the Kohat University of Science and technology, Kohat and the Gomal Medical College, Dera Ismail Khan. The regional ethical committees at both the universities approved the study. The study population was divided into non-COPD controls (N = 123) and patients with COPD (N = 179). Based on Global Initiative for Obstructive Lung Disease (GOLD) classification, the COPD group was further subdivided into two subgroups of mild (GOLD stages 1 & 2; N = 114) and advanced (GOLD stages 3 & 4; N = 65). COPD was defined as the FEV1% / forced vital capacity (FVC) < 0.7 according to the GOLD guidelines (18). All participants were male, 60–65 years of age, with complete data from clinical examination, laboratory investigation, spirometry and HGS measurements. All participants provided written informed consents before participating in the study. The study was conducted in accordance with the declaration of Helsinki (19).
HGS and pedometer
HGS was measured by a digital handgrip dynamometer (CAMRY, South El Monte, CA, USA). The participants were instructed to sit down with their elbows flexed at an angle of 90◦ and the dynamometer in hand in supine position. Three attempts were performed with each hand with a 60-seconds rest between each attempt and the highest value was recorded for analysis. The reference values for the general population were taken from two different meta-analysis (20, 21). The Fitbit one, a commercially available accelerometer-based activity tracker, was used for steps counting for preceding one month before HGS was measured. The participants were asked to wear Fitbit tracker throughout the month and average steps count per day was calculated.
Spirometry
The FEV1, FVC and peak expiratory flow rate (PEFR) were measured using a portable spirometer (Contec SP10, China), according to standards set by American Thoracic Society (22). The participants were instructed to inhale maximally until the lungs were full, followed by forceful exhalation into the spirometer until no air could be exhaled (23). This was done for a minimum of three times and the severity grading was based on FEV1% of predicted values according to the global initiative for chronic obstructive lung disease (GOLD) document, GOLD 1–4 (24).
Measurement of serum biomarkers
Blood samples were drawn in the morning after 10–12 hours of fasting to determine serum metabolic parameters including blood glucose and the high-density lipoproteins—cholesterol (HDL-C) using CardioCheck® equipment (Maxglobal SA, Parsippany, NJ, USA).For analysis of serum biomarkers, 14–18 participants from the non-COPD and each of the two subgroups of COPD participants were selected and the blood samples were drawn in the morning after 10–12 hours of fasting. Serum was assayed using ELISA kits for total sialic acid (abcam, cat # ab83375) and α–1 acid glycoprotein (abcam, cat # ab108852) according to manufacturer’s instructions.
Muscle biopsies
The muscle biopsies were taken from vastus lateralis muscles of selected participants (N = 6—8 per group) using conchotome biopsy method as described previously (25). One part of biopsy specimen was snap frozen and stored at –80◦C while remaining parts were processed for immunostaining and mitochondrial assays.
Protein preparation and western blot
Muscle tissues were homogenized in RIPA buffer containing 50mM Tris (pH = 7.4), 150mM NaCl and protease inhibitors. Proteins were quantified using the Bio-Rad kit (Sigma-Aldrich, Poole, UK) and transferred to nitrocellulose membrane after electrophoresis using 8–15% polyacrylamide gels. Membranes were probed using primary antibodies at 1:1000 dilution each and secondary antibodies at 1:10,000 (HRP-linked anti-rabbit IgG; Cat # 7074S, cell signaling) or 1:25,000 (HRP-linked anti-mouse IgG Cat # 7076S, cell signaling).Primary antibodies used were GRP94 (Cat # 20292T, cell signaling), BIP (Cat # 3177T, cell signaling), CHOP (Cat # 2895T, cell signaling), ATF6 (Cat # 65880, cell signaling), p-NFkB(Cat # 3033, cell signaling), t-NFkB (Cat # 8242, cell signaling), LC3B (Cat # 2775, cell signaling), MyoD (Cat # 13812, cell signaling), p62 (Cat # 5114, cell signaling), p-AkT (Cat # 9271, cell signaling), t-AkT (Cat # 9272, cell signaling), p-P70S6k (Cat # 9205, cell signaling), t-P70S6k (Cat # 9202, cell signaling), p–4EBP1 (Cat # 2855, cell signaling), t–4EBP1 (Cat # 9644, cell signaling), SERCA2 (Cat # 9580, cell signaling), calsequestrin (Cat # ab3516, abcam), DHPR1a (Cat # MA3–920, Thermo Scientific). All image intensities were normalized to protein intensity based on ponceau stain.
Analysis of single fiber diameter, nuclei count and myonuclear domain size
The bundles of ≈80–100 muscle fibers were dissected from fresh tissues and chemically skinned in a solution containing 50% (v/v) glycerol and phosphate buffer saline (PBS) for 24 hours at 4◦C and stored at –80◦C before use, as described previously (26).On the day of experiment, single fiber segments were carefully dissected from the bundle and gently placed on the glass slide. After a brief permeabilization with 0.1% triton X–100 in PBS, fibers were stained with rhodamine-phalloidin (1:200, Molecular Probes Inc, Eugene, OR, USA) for 35 minutes and DAPI (Invitrogen, Molecular Probes, OR, USA) for 5 minutes before final wash. Linear fiber segments of ≈400 µm were chosen for imaging. All images were scanned using LSM 510 Meta confocal microscope and imported into Image J software (Wayne Rasband, National Institute of Health, Bethesda, MD) for analysis.
Sarcoplasmic reticulum Ca2+ ATPase (SERCA) activity assay
The measurement of SERCA ATPase activity was performed in the muscle homogenates at 37◦C using a spectrophotometric assay as described previously (27, 28). Briefly, ≈20mg of muscle samples were diluted 1:10 (w/v) and manually homogenized in ice-cold homogenizing buffer (pH = 7.5) containing (in mM) 250 sucrose, 5 HEPES, 0.2 PMSF and 0.2% NaN3. Ca2+ -dependent ATPase activity was measured in the assay buffer (pH = 7) containing (in mM) 100 KCl, 20 HEPES, 10 MgCl2, 10 NaN3, 10 phosphoenolpyruvate, 1 EGTA, 5 ATP 1 Ca2+ ionophore A–23187 (C–7522, Sigma) and 18 U/ml of both lactate dehydrogenase and pyruvate kinase. The reaction was carried out at 10 different Ca2+ concentrations, ranging between 7.6 and 5 pCa units and was started by adding 0.3 mM NADH. Basal activity was determined in the presence of 40uM of the Ca2+-ATPase inhibitor cyclopiazonic acid (C–1530, Sigma) in dimethyl sulfoxide.
Real-time PCR
Total RNA was extracted from the muscle tissues using TRI reagent (Life technologies, Grand Island, NY, USA). RNA purity and yield were determined by measuring the absorbance at 260 and 280nm. cDNA was prepared from 1mg of the total RNA using iScriptTM cDNA synthesis kit (Bio-Rad, Hercules, CA, USA); 2.5 ng of cDNA samples were amplified using primers for the markers of elevated SR stress (GRP94, BIP& PID), activation of IRE–1α pathway (spliced vs. unspliced XBP1, Deril1, Pdia6), activation of Perk pathway (Perk, ATF4 & CHOP), activation of ATF6 pathway (ATF6α, Hyou1 & Erolib) and 18S along with fast SYBR green master mix (Applied Biosystems, Grand Island, NY, USA). The data was analyzed using the ∆∆Ct method.
Myosin heavy chain (MHC) quantification
The MHC contents were quantified using a high salt buffer as described previously (28). Briefly, 0.6 ug protein was loaded/lane into a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and run at 200 V for 50 min at 4◦C. The gel was stained with coomassie blue, and the optical densities of the bands corresponding to MHC and actin were quantified.
Analysis of F2-isoprostanes
Muscle F2-isoprostane content was measured using thin layer chromatography and mass spectrometry as described before (29). Briefly, ≈150 mg of muscle tissue was homogenized in 10 ml of ice-cold Folch solution (CHC3:MeOH; 2:1) containing butylated hydroxytoluene. Following 30 min incubation at room temperature, 2 ml of 0.9% NaCl was added. The homogenate was centrifuged at 4000g for 5 min at 4◦C. The aqueous layer was discarded while the organic layer was secured for the measurement of esterified F2-isoprostane.
Mitochondrial function
Freshly isolated mitochondria were used for the H2O2 production assay using the Amplex red-HRP method, as described previously (27). The H2O2 dependent oxidation of nonfluorescent Amplex red to fluorescent resorufin was quantified as a measure of mitochondrial H2O2 production. All assays were performed at 37◦C in 96-well plate.
Statistical analysis
The anthropometric characteristics of the participants were presented using mean and standard error of mean as data met the assumption for normality. Student’s t-test or analysis of variance was used to compare continuous variables and the comparison between different groups. A p-value < 0.05was considered to be statistically significant.