Skeletal muscle effects of electrostimulation after COPD exacerbation: a pilot study

Patients

Of the 47 patients with COPD exacerbation who were initially screened, only 17 were enrolled in the study (fig. S1, online supplementary material). 19 were not included due to the presence of one or more exclusion criteria: unstable ischaemic heart disease (n=2), BMI >30 kg·m⁻² (n=5), lung cancer (n=4), associated orthopaedic problems (n=3), scheduled surgery (n=5) or inclusion in another protocol (n=1). 11 patients declined to participate, citing “fear of biopsy” (n=3), “returning home” (n=6), and family refusal (n=1). Among the 17 included patients, one was later excluded due to readmission to ICU following a new exacerbation and another withdrew consent, 48 h after inclusion, owing to family refusal. Finally, nine patients in the NMES group and six in the sham group completed the study. All patients received standard treatment, including nebulised bronchodilators, oral or intravenous antibiotics and oral corticosteroids (30–40 mg daily) for 1–2 weeks. At admission to the ICU, patients were placed under oxygen (three NMES and two sham patients), noninvasive ventilation (three NMES and two sham patients) for 17 days and 20 days, for NMES and sham patients, respectively, or oro-tracheal intubation with mechanical ventilation (three NMES and two sham patients) for 12 days and 11 days, for NMES and sham patients, respectively. The patients' baseline characteristics (table 1) were similar in the two groups. The NMES programme began on average at 12±8 days of hospitalisation in the respiratory ICU.

Tolerance of the NMES sessions

No adverse effect was recorded during the study. All patients could complete 29±1 sessions on average. In the NMES group the intensity of the stimulation was 22±11 mA, 15±5 at inclusion and 47±8 mA and 32±1 mA at the end for quadriceps and hamstring muscles, respectively.

Functional effects of the NMES

MVC changes were statistically different between groups (p=0.02) (fig. 1a), with a clinically significant increase in the NMES group (median (interquartile range) 10 (4.7–11.5) kg; p=0.01) between inclusion and week 6 (fig. S2A, online supplementary material), whereas only a slight and not significant increase was observed in the sham group (median 3 (1–5) kg; p=0.12). Changes in the 6-min walking distance were significant between groups (p=0.008) (fig. 1b), with a clinically significant increase in the distance covered by the NMES group (median 165 (125–203) m; p=0.03) and only a smaller and not significant improvement in the sham group (median 58 (43–115) m; p=0.24) (fig. S2B, online supplementary material). Finally, the dyspnoea score decreased by two points in the NMES group and by only one point in the sham group.

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Figure 1–

Clinical effects of the 6-week-long neuromuscular electrostimulation (NMES) programme. Box plots express changes in a) maximal voluntary contraction (MVC) and b) 6-min walking distance (6MWD), between inclusion and the end of the programme. Standard box plots with medians (interquartile ranges) are depicted. These changes were statistically different between groups. ^#: p=0.02; ^¶: p=0.008.

Muscle oxidative stress

As shown in figure 2a, the anti-DNP antibody detected several protein bands in the quadriceps muscles from patients in the sham and NMES groups. At baseline, the carbonylation levels of total proteins and MHC were comparable in the two groups (figs S3A and S3B, online supplementary material). At the end of the programme, the changes in carbonyl group formation in total proteins and MHC were significantly different between groups (p=0.02 and p=0.04, respectively) (fig. 2b and c). Indeed, carbonylation was significantly decreased in the NMES group when compared to baseline values (p=0.02 and p=0.01, respectively), whereas it remained unchanged in the sham group (p=0.21 and p=0.62, respectively) (figs S3A and S3B, online supplementary material). The carbonylated MHC/total MHC content ratio was also significantly reduced in the NMES group (p=0.03) at the end of the trial, but did not change in the sham group (p=0.40). Although the difference in carbonylated MHC/total MHC content ratio were not significantly different between groups, the effect size tended to be large (effect size 1.09; p=0.08).

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Figure 2–

Protein carbonylation. a) Representative examples of carbonylated proteins in the vastus lateralis muscle from patients in the sham and neuromuscular electrostimulation (NMES) groups, before (B) and after (A) the programme. Carbonylated proteins of different molecular weights were detected. Quantification of the changes in carbonylation (detected as variations in optical density) of b) total proteins and of c) myosin heavy chain (MHC) in the sham and NMES groups. Data are described using standard box plots with medians (interquartile ranges). Total protein and MHC carbonylation changes were significantly different in the two groups, at the end of programme. ^#: p=0.02; ^¶: p=0.04.

The level of 4-HNE protein adducts and lipid peroxidation (data not shown) did not differ between groups at baseline and remained unmodified after the programme (fig. S4A and S4B, online supplementary material). However, the effect size for 4-HNE protein levels tended to be medium (effect size 0.4; p=0.1), that could be considered as a moderate effect.

The levels of Cu/Zn-superoxide dismutase, glutathione reductase and catalase antioxidant enzymes did not significantly change in either group (fig. 3). The effect size for the antioxidant enzymes levels was not calculated because all p-values were >0.16.

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Figure 3–

Antioxidant enzymes. a) Representative examples of glutathione reductase (GR), catalase (CAT), Cu/Zn superoxide dismutase (Cu/Zn-SOD) and GAPDH expression in vastus lateralis from patients in the sham and neuromuscular electrostimulation (NMES) groups, before (B) and after (A) the programme. GAPDH was used as a loading control. Quantification of the change in b) Cu/Zn-SOD, c) CAT and d) GR expression. Data are described using standard box plots with medians (interquartile ranges). Changes in the expression of antioxidant enzymes did not differ in the two groups. ^#: p=0.20; ^¶: p=0.92; ⁺: p=0.55.

Muscle structure

No significant differences were found at baseline between groups (table 2). Changes in the proportion of type I and IIa/IIx fibres were significantly different between groups (p=0.03 and p=0.04, respectively) at the end of the study. The proportion of type I and IIa/IIx fibres significantly increased (from 13±4% to 25±3% (p=0.002) and 9±2% to 23±4% (p=0.003), respectively) in the NMES group, whereas they remained unchanged in the sham group (table 2). A significant negative correlation was observed between changes in the proportion of type I fibres and variation of carbonylated MHC (r= -0.60; p=0.03) (fig. 4a). Although the changes in the proportion of IIx fibres were not significantly different between groups, a significant decrease in the proportion of type IIx fibres in both groups was observed (table 2).

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Figure 4–

Muscle structure. a) The changes in carbonylated myosin heavy chain (MHC) levels, expressed as optical density in arbitrary units, were inversely correlated with the changes in the proportion of type I fibres. b) The increase in total MHC content, expressed as optical densities in arbitrary units, was positively correlated with improvement in the quadriceps maximal voluntary contraction (MVC). •: sham group; ○: neuromuscular electrostimulation patient group.

The size of type I but not of type IIx fibres was significantly increased in the NMES group at the end of the programme (p=0.009 and p=0.16, respectively), whereas it remained unchanged in the control group (data not shown).

Finally, the changes in total MHC content were statistically different between groups (p=0.03). Moreover, a significant positive correlation was found between changes in MHC content and changes in quadriceps strength (r=0.79, p<0.001) (fig. 4b).

Functional effects of the NMES programme

The NMES group presented a more important and significant increase of quadriceps strength that the sham group at the end of the 6-week programme. Although the results were not significant, a slight spontaneous recovery of quadriceps strength occurred in the sham group, which was probably due to the reversibility of the systemic effects of acute COPD exacerbation and the daily mobilisation by a physiotherapist. This result is consistent with the study by Spruit et al. [3]. These authors reported that, 90 days after hospitalisation, the quadriceps peak torque was only increased by a mean of 8% in comparison to the value at day 8 after exacerbation.

The distance covered during the walking test significantly increased in the NMES group, whereas in the sham group the increase was small at the end of the 6-week programme. This result is consistent with the study by Man et al. [17] who have shown that, 3 months after exacerbation, patients in the group of usual care had decreased exercise capacity. The meta-analysis by Puhan et al. [13] has highlighted a significantly improved exercise capacity after early rehabilitation. A recent interesting study showed that outpatient pulmonary rehabilitation immediately following an acute COPD exacerbation improves exercise capacity partly through increase of quadriceps strength [18].

Moreover, the MRC dyspnoea score already started to decrease at the beginning of the programme in the NMES group, concurrently with the improvement in the walking test. Finally, in our study, we did not report the influence of NMES on the respiratory system, but other authors have observed decreased resting respiratory rate in bed-ridden patients, after NMES training [21] and a small but significant reduction in the ventilation dead space was found at peak exercise in treated individuals in comparison with the sham group [19]. These preliminary findings about the possible impact of NMES on the ventilatory response to exercise require confirmation.

In summary, the NMES programme seems to be an effective strategy to facilitate muscle function recovery in COPD patients following ICU hospitalisation for exacerbation. Nevertheless, the underlying molecular mechanisms of such clinical and physiological improvements remain to be elucidated.

Muscle redox balance

Our programme significantly decreased the level of carbonylated proteins in muscle of the NMES group in comparison to the sham group and did not increase the level of 4-HNE protein adducts and lipid peroxidation. This suggests that localised electrostimulation produces muscle contractions of sufficient intensity to induce functional improvement without enhancing oxidative stress within the muscle. This is an important finding, since patients with COPD exhibit increased muscle oxidative stress, not only after high intensity training [10], but also at rest [8]. The 6-week NMES induced, in particular, a decrease of MHC carbonylation. Marin-Corral et al. [30] reported that, in the diaphragm of severe COPD patients, highly carbonylated MHC is likely to be degraded by the ubiquitin–proteasome pathway. Consequently, although the decrease of carbonylated MHC and increase in MHC content in our patients could not be causally related, it is tempting to suggest that the reduction in carbonylated MHC could permit, at least partly, the increase of MHC content. Conversely, it has been proposed that MHC oxidation by reactive oxygen species may perturb myosin structure and impair myosin function [12]. Accordingly, we observed a correlation between changes in total MHC content and changes in quadriceps strength (r=0.79, p<0.001). Altogether, our results suggest that NMES increases muscle strength, probably due to a reduction of the level of MHC oxidation and, therefore, by increasing the quantity of functional MHC in skeletal muscle. However, future work should explore the exact contribution of oxidative stress to the increase in muscle protein breakdown and myosin loss observed in the quadriceps of COPD patients.

Muscle structure

Our study is the first to report changes of muscle fibre types in patients with severe COPD after NMES. Consistent with the results obtained in healthy males [31], paraplegic males [32] and chronic heart failure patients [33] after an electrostimulation programme, we observed an increase in type I fibre proportion and a decrease in type IIx fibre proportion in COPD patients after exacerbation. These changes are usually interpreted as transition from fast-to-slow fibre type. Interestingly, we observed a significant correlation between the changes of type I fibre proportion and of carbonylated MHC level (p=0.03, r= -0.60). It is thus tempting to suggest that the gradual shift in the content of MHC isoforms in COPD patients might be due to a decrease of MHC-I carbonylation and, consequently, of its degradation.

However, the signalling pathways involved in the control of fibre type transition are still unknown. Recently, peroxisome proliferator-activated receptor-coactivator-1 (PGC-1α) was implicated in the formation of oxidative muscle fibres in mice [34]. Moreover, it has been reported that in COPD muscles, the proportion of oxidative fibres is reduced [29] and the level of PGC-1α mRNA is lower [35]. Therefore, it would be interesting to test whether the higher amount of type I fibres observed in the NMES group could be induced by up-regulation of PGC-1α.

Study limitations

One possible critique of our study is the small sample size. However, the retrospectively calculated statistical power of the primary outcome (quadriceps force) was of 96% and significant results were found despite the relatively small population. Furthermore, finding patients with COPD exacerbation who accepted to be included in a study that included two muscle biopsies was challenging, but offered the opportunity to study biochemical and morphological changes of muscle over time in the same patients. However, it is necessary to conduct a clinical study with more patients to confirm our results. In the literature, there is only one example of a similar project, but only one biopsy was carried out at the end of the programme [15]. We believe that our study, as the previous study [15], provides new information for the management of unstable COPD patients.

Given our results and all the existing studies [1, 14, 15, 17, 18] on the effects of early rehabilitation on muscle function and exercise capacity in unstable patients, NMES, not requiring any patient cooperation, may be considered as an alternative to the standard training method used during the first days of hospitalisation following exacerbation. However, when the patient becomes more autonomous, the standard training modality will be able to enhance and potentiate the effects of the NMES. Therefore, further studies are needed to define if the combination of NMES and standard training methods in COPD patients after exacerbation may lead to a greater improvement of overall patient clinical functions than standard training alone.

In conclusion, the application of an NMES programme following exacerbation of COPD is an effective strategy to counterbalance the loss of skeletal muscle function. In our patients, it effectively restored muscle function by decreasing oxidative stress and, particularly, MHC oxidation, and by improving MHC content and distribution of type I muscle fibres. We believe that the decrease in muscle oxidative stress, together with the significant changes in muscle fibre typology, may serve as a basis for designing future studies, where electrical stimulation may be used as a muscle training modality in a larger population of unstable COPD patients.