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J Phys Ther Sci. 2016 May; 28(5): 1595–1598.
Published online 2016 May 31. doi:  10.1589/jpts.28.1595
PMCID: PMC4905918

Differences in strength-duration curves of electrical diagnosis by physiotherapists between DJ-1 homozygous knockout and wild-type mice: a randomized controlled pilot trial

Ju-Hyun Kim, PT, PhD,1,a Won-Deok Lee, PT, MS,2,a Mee-Young Kim, PT, PhD,2 Lim-Kyu Lee, PT, PhD,2,3 Byoung-Sun Park, PT, MS,2 Seung-Min Yang, PT, MS,2 Ji-Woong Noh, PT, MS,2 Yong-Sub Shin, PT, MS,2 Jeong-Uk Lee, PT, PhD,4 Taek-Yong Kwak, PhD,5 Tae-Hyun Lee, PhD,6 Jaehong Park, PhD,7 Bokyung Kim, DVM, PhD,8 and Junghwan Kim, PT, PhD9,*

Abstract

[Purpose] Strength-duration (SD) curves are used in electrical diagnosis by physiotherapists to confirm muscle degeneration. However, the usefulness of SD curves in comparing muscle degeneration in DJ-1 homozygous knockout (DJ-1−/−) and wild-type mice (DJ-1+/+) is not yet fully understood. The electrical properties of the gastrocnemius muscles of DJ-1−/− and DJ-1+/+ mice were compared in the current study. [Subjects and Methods] The electrode of an electrical stimulator was applied to the gastrocnemius muscle to measure the rheobase until the response of contractive muscle to electrical stimulation became visible in mice. [Results] The rheobase of DJ-1−/− mice showed a significant increase in a time-dependent manner, compared to that of DJ-1+/+ mice. [Conclusion] These results demonstrate that the DJ-1 protein may be implicated in the regulation of neuromuscular activity of gastrocnemius muscles of mice.

Key words: DJ-1, Strength-duration (SD) curves, Physiotherapist

INTRODUCTION

DJ-1 (~20 kDa), also called PARK7 and CAP-1, is a small conserved protein associated with autosomal-recessive early onset Parkinson’s disease1,2,3). The human DJ-1 protein contains 189 amino acid residues1,2,3) and acts as a redox sensor, which the thiol group of cysteines of DJ-1 can be oxidized to SOH, S2OH, and S3OH4,5,6). It was proposed that DJ-1 may prevent reactive oxygen species (ROS) accumulation by regulating the levels of other antioxidants. Furthermore, DJ-1 has multiple cellular functions such as transcriptional regulation, protease or redox-dependent chaperone activity, apoptosis and sumoylation5,6,7,8), all of which have been shown to play a role in skeletal muscle activity. Therefore, DJ-1 appears to be an important player in skeletal muscle function during the life-span, although its mechanistic roles are yet to be determined. Skeletal muscle atrophy has proven to be a significant orthopedic problem in the area of physical therapy9,10,11). Muscle atrophy has generally been reported that immobilization-induced atrophy, especially of physiotherapeutic area, decreased muscle volume via decrease of contractile proteins, resulting in muscle weakness and disorder of activities of daily living (ADL)9, 10, 12, 13). In our previous study, it was demonstrated that cast-immobilization- and undernutrition-induced atrophy are correlated with each other via DJ-1 protein in skeletal muscle13). Furthermore, electrical properties such as rheobase and chronaxie are important factors in electrodiagnosis by physiotherapists for measurement of muscle degeneration such as total or partial muscle paralysis14, 15). Rheobase is measured as the threshold stimulus current for an active response with a long-duration pulse and chronaxie is the pulse width at twice the rheobase threshold current14, 15). However, the tendency of change in electrical activities on knockout of DJ-1 is not fully understood. Therefore, in the present study, the electrical properties of the gastrocnemius muscle of DJ-1 knockout (DJ-1−/−) and wild-type (DJ-1+/+) mice were compared.

SUBJECTS AND METHODS

Male DJ-1 homozygous knockout (DJ-1−/−, B6.Cg-Park7tm1shn/J, 25–30 g; n=6) and wild-type (DJ-1+/+; n=6) mice with the same background were purchased from Jackson Laboratory13) (Bar Harbor, ME, USA). To confirm DJ-1 gene depletion in mice, the distal tips of tails were obtained from DJ-1−/− and DJ-1+/+ mice. Genotyping using polymerase chain reaction for DJ-1−/− confirmation was performed with the following primers: DJ-1 forward, 5′-GCT GAA ACT GTG CCA TGT GA-3′; DJ-1 reverse, 5′-TGC TAA AGC GCA TGC TCC AGA CT-3′; Mutant Neo, 5′-TGG ATG TGG AAT GTG TGC GAG-3′. The expression of DJ-1 protein was confirmed in aortic strips using western blotting analysis13, 16, 17). Our investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), and all experiments and animal care conformed to the institutional guidelines established by Konkuk University, Korea. The animals were sacrificed using CO2 inhalation and gastrocnemius skeletal muscles were removed rapidly and carefully from the limbs of animals and placed in cold Krebs solution (containing, in mM: NaCl, 118.0; KCl, 4.8; CaCl2, 2.5; MgSO4, 1.2; NaHCO3, 24.9; glucose 10.0; KH2PO4 1.2) with 95% O2 and 5% CO2 mixed gas13). Rheobase and chronaxie were measured at the regions of the gastrocnemius muscles using an electrical stimulator (Duo 500, Gymnauniphy Co., Belgium). A rheobase measurement pad was applied to the regions of the gastrocnemius muscles of animals until the muscle contraction response became visible14, 15). Data have been expressed as the mean ± SEM. The statistical evaluation of data, using GraphPad Prism (GraphPad Software, San Diego, CA, USA), was performed using Student’s t-tests for group comparisons and ANOVA for multiple comparisons. A p value of <0.05 was considered to be statistically significant.

RESULTS

The rheobase of DJ-1 knockout mice showed a significant increase in a time-dependent manner, compared to that of the wild-type mice (n=6; Table 1).

Table 1.
Differences in the rheobase of DJ-1–/– and DJ-1+/+ mouse gastrocnemius muscles

DISCUSSION

The skeletal muscle is tissue that has high potential plasticity and comprises approximately 40% of the total body weight9, 11). Maintenance of muscle volume and function is important for healthy life, and is important in the rehabilitation of musculoskeletal disease in the field of physical therapy10, 11, 13). However, as muscle mass decreases, there is an accompanying loss of muscle strength and use of nutrients which contributes to reduced muscle function and quality of ADL18,19,20,21). Previous studies and our reports using a model of disuse atrophy induced by cast immobilization indicated loss of muscle mass and cross-sectional area due to a decrease in the rate of protein synthesis9,10,11). The increased degradation of proteins in muscle atrophy is widely coupled with activation of protein ligases such as muscle-specific RING finger-1 (MuRF-1) and the muscle atrophy F-box protein (MAFbx, also called atrogin-1)9,10,11). Our previous data showed that the first to describe the protective functions of DJ-1 against skeletal muscle atrophy are correlated with the MAPK-ubiquitin ligase (both MuRF-1 and atrogin-1) pathway13). In the present study, it was found that DJ-1−/− increased the rheobase in a time-dependent manner. Based on these results, it is cautiously speculated that the increment in the rheobase of DJ-1−/− mice aids in decrease of neuromuscular activities and disorder of movements. This result is similar to that of a previous study that reported that 24-month-old DJ-1−/− mice showed shorter stride lengths than wild-type mice22). Moreover, DJ-1–/– mice exhibit loss of Ca2+ homeostasis, such that decrease in depolarization evoked Ca2+ release from the sarcoplasmic reticulum in the DJ-1 null muscle cells, implying that DJ-1 plays a critical role in calcium regulation of skeletal muscle cells23). However, further systematic and scientific studies in the fields of physical therapy, such as exercise therapy, electrotherapy, neurophysiotherapy, and hydrotherapy, are needed to confirm the mechanisms underlying the effects of DJ-1 in atrophied muscle strips and cells24,25,26,27). In summary, the rheobase of DJ-1 knockout mice was significantly higher than that of wild-type mice. The present results suggest that DJ-1 affects the important play a role in muscle activity13, 22, 23, 26).

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