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Purpose: Skeletal muscle damage occurs following high-intensity or unaccustomed exercise; however, it is difficult to monitor damage to the respiratory muscles, particularly in humans. The aim of this study was to use clinical measures to investigate the presence of skeletal muscle damage in the inspiratory muscles. Methods: Ten healthy subjects underwent 60 minutes of voluntary inspiratory threshold loading (ITL) at 70% of maximal inspiratory pressure. Maximal inspiratory and expiratory mouth pressures, delayed onset muscle soreness on a visual analogue scale and plasma creatine kinase were measured prior to ITL, and at repeated time points after ITL (4, 24 and 48 hours post-ITL). Results: Delayed onset muscle soreness was present in all subjects 24 hours following ITL (intensity = 22 ± 6 mm; significantly higher than baseline p = 0.02). Muscle soreness was reported primarily in the anterior neck region, and was correlated to the amount of work done by the inspiratory muscles during ITL (r = 0.72, p = 0.02). However, no significant change was observed in maximal inspiratory or expiratory pressures or creatine kinase. Conclusions: These findings suggest that an intense bout of ITL results in muscle soreness primarily in the accessory muscles of inspiration, however, may be insufficient to cause significant muscle damage in healthy adults.
High intensity, unaccustomed, or eccentric exercise can induce muscle damage, characterized by the disruption of myofilaments and cytoskeletal elements1,2 as well as an inflammatory response.3,4 In addition to the cellular response, muscle damage is also manifested as a loss of muscle force5,6 and delayed onset muscle soreness (DOMS), peaking 24 to 48 hours following the exercise bout.5,6 Cellular constituents of muscle such as creatine kinase (CK), lactate dehydrogenase, and myosin heavy chain leak into the plasma and can also be used as quantitative markers of skeletal muscle damage.7,8 These markers show a time course of change and recovery that ranges between hours and days.8,9
The majority of studies examining skeletal muscle damage in humans are limited to the limb muscles, commonly the elbow flexors and knee extensors.10 However, the muscles of respiration that are essential to life, may also be prone to exercise-induced damage when faced with high intensity or prolonged loads. In animal studies, muscle damage of the diaphragm has been observed following both short bouts of high inspiratory resistive loading11,12 and prolonged periods of low inspiratory loads.13,14 Diaphragm injury has also been observed in people with chronic obstructive pulmonary disease (COPD) and correlated to the severity of airflow obstruction, demonstrating a relationship between respiratory load and muscle damage.15,16
Inspiratory muscle damage has important clinical implications as it may lead to respiratory failure.12 Inspiratory muscle training, used to wean mechanically ventilated patients17,18 and to improve functional outcomes in people with COPD,19 could be deleterious if prescribed too aggressively. Therefore, it is essential for clinicians to be able to assess the presence of muscle damage when implementing an inspiratory muscle training program, by using outcome measures that are minimally invasive. Delayed onset muscle soreness and muscle force deficits are common clinical measures of muscle damage. Plasma CK is routinely used to diagnose muscle damage in cases of myocardial infarction and rhabdomyolysis,20 therefore widely available in the clinical setting.
We aimed to determine whether there were changes that could be detected in clinical measures, including maximal inspiratory and expiratory pressures (MIP and MEP), DOMS, and plasma CK that may indicate inspiratory muscle damage following a bout of inspiratory loading exercise in healthy adults. We hypothesized that a high inspiratory threshold load would result in increased DOMS, impaired MIP, and increased plasma CK following the inspiratory loading task. A sample of healthy, young adults was chosen for this study to provide initial data on how much damage could be expected and whether this task may be feasible in older adults or people with respiratory disease.
Ten adults [M/F: 5/5; 27 (4) years] were recruited by posting notices at recreational facilities, the university campus, and public libraries. Subjects were excluded if they: (1) had a history of respiratory disease or abnormal pulmonary function, (2) had a diagnosis of cardiovascular or neuromuscular disease, (3) were current or ex-smokers, (4) were pregnant or taking contraceptive agents containing estrogen and/or progesterone, or (5) were competitive or elite athletes. Recreational activity, determined by a brief questionnaire, was acceptable. The study was approved by the University of British Columbia's Clinical Research Ethics Board and all subjects provided written, informed consent.
Subjects were asked to refrain from exercising for at least 2 days prior to baseline measures and throughout the study. Females were tested only during the mid-follicular phase [day 9 (4)] of their menstrual cycle to minimize the confounding effects of estrogen on muscle damage.21 Age, sex, height, weight, FEV1, and FVC were recorded. During 2 baseline sessions held at least 2 days apart, respiratory muscle strength (MIP, MEP), plasma CK, and DOMS were measured. On the third testing day, subjects performed a 60 min bout of inspiratory muscle threshold loading (ITL) targeted at 70% of their MIP obtained at baseline. Post-exercise assessments of DOMS, MIP, and MEP were conducted at: 4, 24, and 48 hours post-ITL. Plasma CK was measured at 24 and 48 hours post-ITL.
Spirometric variables (FVC, FEV1) were measured using standardized procedures22 with a portable Spirolab II (Medical International Research, Vancouver, BC) to confirm normal pulmonary function.
Maximal inspiratory pressure and MEP were performed using standard procedure.22,23 A rubber, flanged mouthpiece was attached to a 3-way stopcock and valve (2100 series, Hans Rudolph, Kansas City, MO) connected to a pressure transducer (MP45-36-871, Validyne, Northridge, CA). The transducer was calibrated prior to each testing session. The signal from the transducer was sampled using an analogue to digital converter (Powerlab ML-880) and collected using Chart software (ADInstruments Inc., Colorado Springs, CO). Participants were seated upright with their back and feet supported. Subjects wore noseclips and were instructed to exhale to residual volume, then to inhale maximally for 5 seconds to obtain MIP. For MEP, subjects were instructed to inhale to total lung capacity, then to expire maximally for 5 seconds. Strong verbal encouragement was provided by the tester. The highest pressure averaged over a one-second interval was recorded as the MIP or MEP. Each subject performed up to 10 trials of MIP and MEP until 3 measurements ranging within 5% were obtained. The average of the 3 highest, reliable trials was taken as the maximal pressure23 and expressed as a percent of predicted.24
Delayed onset muscle soreness was measured with the use of a 100 mm visual analogue scale (VAS)25 and body diagram. The subject was asked to rate the intensity of muscle soreness felt during a MIP by making a mark along a line anchored with the terms “no soreness” (ie, 0) to “worse soreness ever felt” (ie, 100 mm). The subject was also asked to shade in areas on a body diagram to indicate the location of their muscle soreness.
Blood samples (~10 ml) were taken from the medial cubital vein of the forearm and immediately placed in a lithium heparin, PST vacutainer tube (BD-Canada, Ontario). Samples were centrifuged and refrigerated at 4°C for subsequent analysis. Samples were processed for CK concentration using the Flex® assay on an integrated chemistry analyzer (Dimension RxL Max System, Siemens) that examines for levels of muscle isoform of creatine kinase after myocardial infarction, muscle disease, strenuous exercise, and muscle injury. This assay is based on the methods previously described26,27 and updated according to the Clinical and Laboratory Standards Institute.28 The analyzer was calibrated before and after daily runs of samples. The laboratory reference values for this measurement are 35-250 U/L for males and 20-230 U/L for females. The in-house reproducibility of CK assay has a coefficient of variation of 3%.
The subject was seated with feet and back supported, and elbows resting on a table. The threshold loading device29,30 (Figure (Figure2)2) was placed on the table and its height was adjusted so the subject could reach the mouthpiece with his or her head and neck in neutral alignment. The threshold loading device was attached in series with a pneumotach and the inspiratory port of a one-way valve. Adjustable weights attached to a plunger provided an inspiratory threshold load that had to be met in order for the subject to inhale. No airflow occurred unless the threshold pressure was met. Weights corresponding to a threshold pressure of 70% of the subject's MIP (determined from 2 baseline sessions) were attached to the threshold loading device (Figure (Figure2).2). Subjects were given an auditory cue to inspire against the threshold load of 70% MIP for 2 seconds and expire for 4 seconds (duty cycle of 33%), at a rate of 10 breaths per minute. The task was continued for 60 minutes. Verbal encouragement was given at 5 minute intervals. If the subject was not able to meet the threshold load, the weight was dropped to 65% MIP and then to 60% of MIP. All subjects were able to complete the ITL task, maintaining 60% to 70% of MIP for the duration of 60 minutes.
A pneumotach (Hans Rudolph, Kansas City, MO) measured airflow and volume throughout the duration of the task. Mouth pressure was monitored continuously using a pressure transducer attached to the mouthpiece. The total inspiratory force generated over the entire ITL task was estimated for each subject from the product of the breathing frequency and the integral of the mouth pressure (fB × ∫ Pm).31,32 End tidal CO2 was monitored via a CD-3A CO2 analyzer (Ametek, Pittsburgh, PA) and the subject was maintained eucapnic by adding CO2 to the inspired air, if needed. Heart rate and oxygen saturation (504 Pulse Oximeter, Criticare Systems Inc., Waukesha, WI) were also monitored throughout and no changes were observed from resting values in these variables.
Statistical analysis was conducted using Statistical Package for the Social Sciences (version 11.0, SPSS Inc, Chicago, IL). Data from men and women were pooled, as preliminary analysis did not reveal any sex differences. Group means and standard deviations or standard error of the mean, as indicated [mean (SD) or (SEM)] are presented. Repeated measures analysis of variance and Tukey's post-hoc tests were used to test for differences in MIP, MEP, VAS rating of DOMS, and CK level from baseline values. Bivariate correlations between MIP, total inspiratory force, and DOMS were determined using Pearson's product-moment correlation coefficients.
All subjects had normal values for baseline pulmonary function (FEV1, FVC) and baseline MIP and MEP (Table (Table11).
Subjects demonstrated consistent inspiratory muscle force, inspiratory flow rate, and end-tidal CO2 over 60 minutes of ITL (Table (Table2).2). Peak mouth pressure remained, on average, within 10 cmH2O throughout the task for individual subjects. PETCO2 was maintained within 1-5mmHg of resting values and no subjects showed CO2 retention. Raw data tracings of these measures from a representative subject are shown in Figure Figure3.3. The amount of inspiratory force generated over the entire ITL task (fb × ∫ Pm) ranged from 4.90 × 104 to 10.6 × 104 cmH2O/min, and was correlated to the baseline MIP (r = 0.66, p = 0.03; Figure Figure4A4A).
At 4, 24, and 48 hours post-ITL, MIP was 92 (10)%, 96 (12)%, and 103 (10)% [mean (SD)] of baseline respectively, and was not different from baseline values (Figure (Figure5).5). Similarly, no significant differences were observed in MEP from baseline values following ITL.
Nine of 10 subjects reported no DOMS at baseline; however, one subject indicated a baseline soreness of 18 mm on the 100 mm line. All subjects reported an increase in DOMS after ITL that was significantly higher than baseline [mean (SEM) 6 (2) mm] at 4 [22 (8) mm; p = 0.02] and 24 hours [22 (6) mm; p = 0.02]. Regarding location of DOMS, 7 out of 10 subjects reported muscle soreness in the anterior neck region. Other common sites for muscle soreness were the region of the diaphragm (along the lower border of the ribcage, n = 4), parasternal region (n = 3), and the posterior neck (n = 2) (Figure (Figure6A).6A). Delayed onset muscle soreness at 24 hours was correlated to the total inspiratory muscle force generated during the task (r = 0.72, p = 0.02; Figure Figure4B).4B). At 48 hours post-ITL, muscle soreness was significantly lower than at 4 hours post-ITL [11 (4) mm at 48 hours; p = 0.03] and no longer different than baseline values (Figure (Figure6B6B).
Plasma CK was within the normal range in all subjects at baseline [mean (SEM), 79.8 (9.2) U/L] and did not differ at 24 and 48 hours post-ITL [76.8 (8.7) and 81.5 (9.4) U/L, respectively].
The results of this study indicate that high intensity ITL did not appear to induce muscle damage in healthy adults. Although all of the subjects reported an increase in muscle soreness at 24 hours following ITL from baseline, the change was below the threshold for a clinically important difference in the majority of subjects.33 We observed that the majority of subjects reported soreness in the small muscles of the anterior neck, with fewer experiencing soreness in the diaphragm region. This may be attributed to differences in susceptibility of muscle damage between the diaphragm and accessory muscles, different thresholds in the perception of DOMS, or differential loading on these muscles during ITL. Furthermore, DOMS was correlated to the amount of work (or load) placed on the muscles of inspiration during the task indicating that heavier contractile loads resulted in more muscle soreness. The presence of DOMS was not accompanied by other markers of muscle damage such as force deficits or increased plasma CK that supports the presumption that the ITL protocol was not sufficiently intense to cause muscle damage in healthy adults.
We observed that ITL resulted in DOMS in the inspiratory muscles and peaked 24 hours following the ITL bout, which is the typical response to muscle damage.3 However the change in the VAS rating of DOMS from baseline to 24 hours was at or below the minimally clinical important difference (MCID) for the VAS of approximately 17 to 20mm33,34 in our subjects, with only 3 subjects exceeding the MCID. Furthermore DOMS is an indirect marker of muscle damage that has been associated with the inflammatory response occurring as a response to the disruption of muscle fibers.35 Although the magnitude of DOMS may not always reflect the degree of muscle damage,36 we did observe that the intensity of DOMS was associated with the total force generated by the inspiratory muscles. This is in agreement with previous studies in animal models that have shown that the load placed on a skeletal muscle is related to the amount of damage observed both functionally and histologically.37,38 However studies in humans show conflicting results on this relationship39,40 and may be confounded by other factors affecting skeletal muscle damage such as muscle strain, muscle length, or contraction velocity.39
We did not observe other typical markers of muscle damage such as a force deficit or an increase in CK following ITL in our subjects. Both of these measures may have limitations in its sensitivity to detect damage of the inspiratory muscles. Specifically, MIP provides a global estimate of the pressure generated by the diaphragm, other inspiratory muscles, and outward recoil of the chest wall.41 Therefore force deficits in specific inspiratory muscles cannot be extrapolated from this measure. In our study, differential inspiratory muscle recruitment was not monitored during the task due to the limitations in obtaining an accurate signal from surface EMG of the diaphragm.42 However, we hypothesize that there was alternating recruitment of the diaphragm and other inspiratory muscles throughout the ITL task. Previous studies demonstrated diaphragmatic fatigue or task failure with loads of 60% of MIP.43,44 However, Eastwood et al45 reported that when inspiratory muscle recruitment pattern was not constrained to focus on diaphragm recruitment during a progressive ITL task, the onset of muscle fatigue was delayed and maximal inspiratory pressures were maintained after ITL.45 Similarly in our study, subjects could have recruited a combination of muscles during ITL to produce a maximal inspiratory effort, thus a drop in MIP was not detectable following ITL even if certain muscles experienced fiber disruption.
Plasma CK, a commonly used marker of muscle damage,8,20 has been reported to increase following eccentric exercise of muscle groups such as the knee extensors or elbow flexors.46 However, CK does show a highly variable response following eccentric exercise that may limit its sensitivity.8 In the current study, the small total mass of the muscles involved in the ITL task would likely have resulted in only a small increase in CK, which may have been diluted by the circulating blood volume. In addition the ITL task did not target eccentric loading of the diaphragm that may further limit muscle damage and CK release as compared to studies on limb muscle where eccentric contractions are used. In light of these factors, the release of CK from injured muscle fibers may have been difficult to detect using a venous blood sample. Other plasma markers of muscle damage such as myosin heavy chain fragments or skeletal troponin I may be more sensitive to a small amount of muscle damage;47 however, this requires further investigation.
Delayed onset muscle soreness was consistently reported by subjects in the anterior neck region, which is the location of the other muscles of inspiration including the sternomastoid, scalenes, as well as the so-called “strap muscles” (ie, hyoid muscles). Due to their anatomical location, these muscles have a short working range that may put them at greater risk for muscle damage. According to the sarcomere nonuniformity hypothesis,48 shorter muscles that have fewer sarcomeres in series are more susceptible to muscle damage. This is due to a greater number of sarcomeres being placed in a weakened position, along the descending limb of the length-tension curve. These overstretched sarcomeres have little myofilament overlap, and are likely to fail under tension and become disrupted.48 With repeated contractions, the number of disrupted sarcomeres increases and the muscle membrane becomes damaged.48 In our study, ITL may have resulted in muscle disruption of the susceptible muscles of the anterior neck rather than the diaphragm, resulting in more muscle soreness in these muscles.
It is difficult to confirm whether muscle damage occurred within the diaphragm using noninvasive, clinical markers. Clinical measurements such as MIP do not provide specific information about diaphragmatic force generation and DOMS from the diaphragm may have been decreased or referred to upper cervical dermatomes (C3, 4, 5) due to its internal location. Alternatively, the ITL task may have been insufficient to cause damage specifically to the diaphragm. Structural disruption of the sarcomeres within the diaphragm has been reported following a much shorter bout of ITL (approximately 12-17 minutes at 80% of MIP);16 however, no clinical measures of muscle force or soreness were reported in this study. Furthermore the sample of the previous study consisted of older adults and people with COPD; therefore, their subjects may have a different damage response following ITL than our young, healthy group of subjects.49,50
We employed voluntary inspiratory efforts to quantify maximal pressure development before and after the ITL, as well as during the task itself. Therefore, we were unable to isolate a specific muscle such as the diaphragm and the ability to voluntarily recruit the diaphragm may have confounded our ability to detect force deficits following ITL. Phrenic nerve stimulation using magnetic or transcutaneous stimulation has previously been used to directly stimulate the diaphragm muscle43 and may be useful in assessing whether force deficits indicative of muscle damage are present following an intense bout of ITL.
Finally, this study was conducted in a small sample of healthy adults and we achieved statistical power of 0.68 for detecting significant differences in the outcome of MIP and 0.37 for plasma CK, over repeated time points.51 Therefore a larger sample of subjects with similar variability would have been needed to detect statistically significant changes in these variables following ITL. Furthermore a sample of at least 14 subjects would be needed to detect a mean change in VAS of 20 mmHg with repeated time points, given a similar variability as observed in our sample.
An intense bout of ITL resulted in DOMS primarily in the anterior neck muscles of inspiration in a group of young, healthy adults. Other markers of muscle damage (force deficit, plasma CK) did not change with ITL and the increase in muscle soreness at 24 hours was on average smaller than the MCID for the VAS in detecting DOMS, indicating that the subjects did not experience clinically significant muscle damage in their inspiratory muscles. This finding may have important implications for inspiratory muscle training, indicating that intense loading may not result in muscle damage in healthy people and therefore can be safely prescribed. Given that several inspiratory muscles work together to produce inspiratory force, these muscles may be resilient to muscle damage as they can compensate for high loads through differential muscle recruitment. However in patient populations who experience diaphragm weakness such as COPD, the susceptibility to muscle damage may be increased and needs to be examined specifically. Future studies should examine the presence of DOMS and inspiratory force deficits with different doses of ITL (ie, % of MIP and duration of task), as well as in patients performing inspiratory muscle training as a physical therapy intervention.
We thank our subjects for their enthusiastic participation. We would also like to thank Steven Lammers and Jonathan Witt for their contributions to data collection and analysis. SM was a postdoctoral fellow at the University of British Columbia at the time of this study and was supported by a postdoctoral fellowship from the British Columbia Lung Association. AWS was supported by a Scholar Award from the Michael Smith Foundation for Health Research, a New Investigator Award from the Canadian Institutes of Health Research and in part, by the Natural Sciences and Engineering Research Council of Canada.