Methodological considerations for investigating the influence of neuromuscular electrical stimulation on pH heterogeneity

We read with interest the study by Stutzig et al (1) investigating the influence of neuromuscular electrical stimulation (NMES) on pH heterogeneity. The authors reported a threefold splitting of the Pi peak in response to NMES and concluded that the three corresponding pH values represent the three different fiber types (I, IIa, and IIx). We would like to raise several issues about the novelty of the present findings as well as the corresponding physiological interpretations. In a recent study, use of an advanced P chemical shift imaging methodology revealed the occurrence of two peaks of Pi in 13 of 15 subjects in response to NMES that was applied to the vastus lateralis muscle (2). In addition, the occurrence of Pi splitting was higher for NMES than for a voluntary protocol performed at the same force levels. It has been suggested that the pH values associated with each Pi peak during NMES could be representative of two different populations of exercising muscle fibers (ie, glycolytic versus oxidative), thereby demonstrating the specificity of motor unit recruitment associated with NMES (3). Although Stutzig et al observed a phosphocreatine depletion similar to that reported previously (2), the authors differentiated three peaks of Pi in all of their subjects. Although briefly mentioned in the Discussion, it must be noted that the authors used a nonlocalized acquisition scheme that has methodological limitations. Indeed, Meyerspeer et al (4) elegantly demonstrated that Pi splitting during exercise occurred only with nonlocalized P measurements, whereas this phenomenon was absent when using a localized acquisition scheme. One could therefore assume that the alkaline peak of Pi detected by the authors reflected a signal contamination from the stimulation of the neighboring soleus and gastrocnemius lateralis muscles (illustrated by the changes in T 2-weighted images), inasmuch as no significant difference was observed between the resting pH and the alkaline pH. It would have been of interest to estimate the contribution of these two nondirectly stimulated muscles relative to the P signals to rule out any potential signal contamination. Furthermore, it turns out that there was a large interindividual variability in the pH alkaline values, with two outliers showing a pH of 6.3 and 6.4 (Fig. 7). It is unclear whether this variability could be related to a difference in training status (eg, sedentary versus active) among the subjects. Furthermore, it remains to be determined whether the signal-to-noise ratio was high enough to accurately and reliably separate the three Pi resonances. A close visual inspection of the adenosine triphosphate signals from the individual dataset (Supporting Figs. S3 and S5) raises a concern about the sensitivity of the measurements. Unfortunately, the reliability of the present P analysis was not reported. Considering the aforementioned methodological issues associated with nonlocalized P measurements, caution is required when interpreting pH heterogeneity in relation to muscle fiber type composition. We agree that NMES is the method of choice for investigating pH heterogeneity in stimulated muscles. However, we believe strongly that the use of more accurate localized P-MRS techniques (eg, semi-LASER) at higher magnetic fields (eg, 7T) (4), which are associated with negligible voxel contamination (ie, 1%) and high temporal resolution (eg, 6 s), would provide relevant information about the physiological mechanisms accounting for the occurrence of Pi splitting during NMES.