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Impaired post-tetanic potentiation of muscle twitch in myasthenia gravis
Clinical Neurophysiology 127 (2016) 1689–1693
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Clinical Neurophysiology journal homepage: www.elsevier.com/locate/clinph
Impaired post-tetanic potentiation of muscle twitch in myasthenia gravis Daisuke Yamamoto a, Tomihiro Imai b,⇑, Emiko Tsuda a, Takayoshi Hozuki c, Rika Yamauchi d, Shin Hisahara a, Jun Kawamata a, Shun Shimohama a a
Department of Neurology, Sapporo Medical University School of Medicine, Sapporo, Japan Department of Occupational Therapy, Sapporo Medical University School of Health Sciences, Sapporo, Japan Sapporo Shirakabadai Hospital, Sapporo, Japan d Department of Neurology, Sunagawa City Medical Center, Sunagawa, Japan b c
a r t i c l e
i n f o
Article history: Accepted 10 October 2015 Available online 23 October 2015 Keywords: Myasthenia gravis Muscle fatigue Staircase phenomenon Excitation–contraction coupling Post-tetanic potentiation
h i g h l i g h t s A novel method using an accelerometer was used to evaluate post-tetanic potentiation of muscle
twitch in myasthenia gravis. Significant decrease in post-tetanic potentiation of muscle twitch may be associated with impairment
of excitation–contraction coupling in myasthenia gravis. The present method has high sensitivity in detecting impairment of excitation–contraction coupling
in myasthenia gravis.
a b s t r a c t Objective: The aim of this study was to evaluate post-tetanic potentiation of muscle twitch in myasthenia gravis (MG). Methods: Post-tetanic potentiation was evaluated by recording the compound muscle action potential (CMAP) of abductor pollicis brevis and movement-related potential (MRP) of the thumb using an accelerometer after tetanic stimulation of the median nerve at the wrist. After baseline recording, tetanic stimulation was delivered to the median nerve at a frequency of 10 Hz for 10 s. The CMAP and MRP were successively recorded at baseline and at 5, 10, 30, 60, 90 and 120 s after tetanic stimulation. The chronological changes of CMAPs and MRPs were recorded bilaterally in 11 patients with MG, 9 patients with myopathies (disease controls), and 25 healthy control subjects. Results: Maximal acceleration of MRP was significantly elevated during 10 s after tetanic stimulation without any CMAP changes in all groups. However, statistical analysis detected a significant decrease in post-tetanic potentiation of maximal acceleration of MRP in MG patients only compared to healthy controls, but not in myopathy patients, which may imply impairment of excitation–contraction coupling in MG. Conclusions: Post-tetanic potentiation of muscle twitch is significantly diminished in MG, suggesting impaired excitation–contraction coupling. Significance: Measurement of post-tetanic potentiation using an accelerometer is a simple and sensitive method to detect impairment of excitation–contraction coupling in MG. Ó 2015 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction
⇑ Corresponding author at: Department of Occupational Therapy, Sapporo Medical University School of Health Sciences, South 1, West 17, Chuo-ku, Sapporo 060-8556, Japan. Tel.: +81 11 611 2111; fax: +81 11 611 2115. E-mail address: [email protected] (T. Imai).
Although defective neuromuscular transmission is known to be a cause of muscle weakness in myasthenia gravis (MG), previous studies have shown the possible roles of other processes, the failure of which may also cause muscle weakness in MG. Especially,
http://dx.doi.org/10.1016/j.clinph.2015.10.037 1388-2457/Ó 2015 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.
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excitation–contraction coupling may be defective in patients with MG (Pagala et al., 1990, 1993; Slomic´ et al., 1968). The excitation– contraction coupling in skeletal muscle is the process whereby an action potential triggers a muscle fiber to contract. Some investigators have applied the post-tetanic potentiation and/or the staircase phenomenon to elucidate the impairment of excitation–contraction coupling in MG (Krarup, 1977; Slomic´ et al., 1968). Posttetanic potentiation is the enhancement of active twitch force following high-frequency tetanic stimulation (Brown and von Euler, 1938), while the staircase phenomenon is the progressive increase in active twitch force during repetitive low-frequency stimulation (Bowditch, 1870/71). The post-tetanic potentiation and staircase phenomenon have been explained by an increase in sarcoplasmic Ca2+, which may induce facilitation of excitation–contraction coupling after tetanic stimulation and stepwise increase in response during repetitive twitch, respectively. Studies in MG patients have demonstrated a correlation between the magnitude of potentiation and the severity of MG, and proposed the significance of impaired excitation–contraction coupling in MG (Krarup, 1977; Slomic´ et al., 1968). However, post-tetanic potentiation and staircase phenomenon are rarely used to evaluate the impairment of excitation–contraction coupling in the clinical laboratory, presumably because complicated apparatus using a strain gauge is required. In this study, we describe a novel procedure to measure posttetanic potentiation using an accelerometer attached to the hand muscle instead of the complicated apparatus with a strain gauge used in previous studies. Our method is simple and may be a useful clinical test for the detection of impairment of excitation–contraction coupling in MG.
2. Materials and methods 2.1. Subjects and protocol We studied 11 patients with MG (3 males and 8 females; aged 33–67 years, mean 47.2 years) at Sapporo Medical University
Hospital. The diagnosis of MG was based on typical clinical features and electrophysiological evidence of a defect in neuromuscular transmission, which is either an abnormal decrement in repetitive nerve stimulation tests (muscles tested: orbicularis oculi, nasalis, trapezius, abductor pollicis brevis and abductor digiti minimi), or increased jitter on concentric needle single fiber electromyography of the voluntarily activated extensor digitorum communis and orbicularis oculi muscles (Kokubun et al., 2012; Kouyoumdjian and Stålberg, 2008). Acetylcholine receptor (AChR) binding antibody was measured in a commercial laboratory using a radioimmunoassay. In MG patients without AChR antibody, serum antibody against muscle-specific receptor tyrosine kinase (MuSK) was measured by immunoprecipitation of 125I-recombinant MuSK extracellular domains (Shiraishi et al., 2005). The diagnosis of thymic lesion was based on histopathological findings after extended thymectomy. At the time of the present study, 1 of 11 patients had never received any specific MG therapy, and the remaining 10 patients showed exacerbation of myasthenic symptoms despite receiving corticosteroids alone (5 patients), or corticosteroids combined with tacrolimus (4 patients) or cyclosporine (1 patient). Extended thymectomy had already been performed in 8 patients. Disease severity was graded according to the clinical classification of Myasthenia Gravis Foundation of America (MGFA) (Jaretzki et al., 2000). We also studied 25 healthy control subjects (9 males and 16 females) aged 26–60 years (mean, 40.2 years) to establish the normal ranges for the methods used in this study, and 9 patients with myopathies (3 males and 6 females) aged 21–66 years (mean, 48.4 years) to examine whether myopathic changes influence excitation–contraction coupling (Table 1). Myopathies included autoimmune myositis, muscular dystrophy and muscle sarcoidosis. All the healthy control subjects and patients gave informed consent for participation in this study. The study was approved by the Ethics Committee, Sapporo Medical University Hospital, Sapporo, Japan.
Table 1 Comparison of age, grip power and electrophysiological findings in subject groups. Subject group
Number of subjects (males, females) Age (years) Number of hands tested
F value
Healthy control
Myasthenia gravis
Myopathy
25 (9, 16) 40.2 (11.3, 27–60) 50
11 (2, 9) 47.2 (12.8, 33–67) 22
9 (3, 6) 48.4 (14.0, 21–66) 18
Grip power (kg)
Lt + Rt Lt Rt
32.4 (8.5, 21–54) 31.0 (7.5, 21–46) 33.7 (9.4, 23–54)
19.2 (10.3, 5–48)⁄⁄ 18.9 (9.6, 10–43)⁄⁄ 19.5 (11.3, 5–48)⁄⁄
16.4 (8.8, 3.5–35)⁄⁄ 15.4 (9.4, 3.5–32)⁄⁄ 17.4 (8.7, 5–35)⁄⁄
0.09 0.25 0.38
CMAP amplitude (mV)
Lt + Rt Lt Rt
10.6 (1.9, 6.9–13.8) 11.0 (1.7, 7.0–13.5) 10.3 (2.0, 6.9–13.8)
8.0 (2.7, 3.5–14.3)⁄⁄ 7.9 (2.5, 4.1–11.2)⁄⁄ 8.2 (3.0, 3.5–14.3)
9.1 (3.4, 1.5–15.1) 9.3 (3.3, 3.1–13.3) 8.8 (3.8, 1.5–15.1)
4.60 3.00 2.16
CMAP area (msmV)
Lt + Rt Lt Rt
30.0 (5.7, 17.7–42.6) 30.5 (5.2, 22.5–42.6) 29.4 (6.2, 17.7–42.1)
22.2 (7.9, 10.2–39.8)⁄⁄ 22.9 (9.1, 10.3–39.8)⁄ 21.5 (7.0, 10.2–35.3)⁄⁄
25.9 (10.5, 4.0–42.4) 26.5 (11.0, 6.8–42.4) 25.3 (10.7, 4.0–40.2)
5.12 4.67 1.32
Maximal acceleration at baseline (m/s2)
Lt + Rt Lt Rt
5.5 (3.8, 1.6–17.6) 5.6 (4.0, 1.8–17.6) 5.4 (3.6, 1.6–14.7)
2.9 (1.0, 0.8–5.3)⁄⁄ 2.9 (0.9, 1.6–4.0)⁄⁄ 2.8 (1.2, 0.8–5.3)
3.2 (1.6, 0.6–7.4)⁄ 3.2 (1.4, 0.6–5.1) 3.2 (1.8, 1.2–7.4)
8.64 3.26 6.06
Maximal acceleration at 5 s after TS (m/s2)
Lt + Rt Lt Rt
9.5 (7.2, 2.7–36.0) 9.9 (7.9, 3.1–36.0) 9.0 (6.6, 2.7–28.1)
4.2 (1.7, 1.1–8.1)⁄⁄ 4.3 (1.6, 2.1–6.5)⁄⁄ 4.0 (1.8, 1.1–8.1)⁄
5.3 (2.7, 1.0–10.3)⁄ 5.4 (2.9, 1.0–9.5) 5.2 (2.7, 1.7–10.3)
8.99 3.57 6.17
PTP of MRP acceleration (5 s after TS divided by baseline)
Lt + Rt Lt Rt
1.7 (0.4, 1.0–2.6) 1.8 (0.4, 1.2–2.6) 1.7 (0.3, 1.0–2.2)
1.5 (0.2, 1.0–2.0)⁄⁄ 1.4 (0.2, 1.2–1.9) 1.5 (0.3, 1.0–2.0)
1.7 (0.4, 1.0–2.2) 1.7 (0.4, 1.0–2.2) 1.6 (0.3, 1.1–2.1)
1.56 1.97 0.16
Data are expressed as mean (SD, range) for age, grip power, CMAP amplitude, CMAP area, ECCT, and acceleration parameters. Single asterisk and double asterisks denote p < 0.05 and p < 0.01, respectively, compared to healthy control values, as analyzed by Mann–Whitney U-test. CMAP, compound muscle action potential; MRP, movementrelated potential; ECCT, excitation–contraction coupling time; TS, tetanic stimulation; PTP, post-tetanic potentiation, Lt, left hand; Rt, right hand.
D. Yamamoto et al. / Clinical Neurophysiology 127 (2016) 1689–1693
2.2. Measurement of baseline compound muscle action potentials and movement-related potentials Compound muscle action potentials (CMAPs) were recorded from the abductor pollicis brevis using surface disc electrodes in belly-tendon arrangement after median nerve stimulation at the wrist. Simultaneously, movement-related potentials (MRPs) were recorded using an accelerometer (SV1101, NEC, Japan) taped to the tip of the thumb (Nakata et al., 2007). The amplitude and area of CMAP were measured from the baseline to the negative peak using a cursor. The maximal acceleration of thumb movement was obtained from the initial peak amplitude of MRP. For stimulation, a 0.2-ms rectangular pulse was delivered to the median nerve at the wrist with gradually increasing intensity to reach a supramaximal response. Once a supramaximal CMAP was obtained, the initial recording of resting CMAP and MRP was performed (baseline). All stimulating and recording procedures were performed using an electromyograph (Nicolet Viking IV, Nicolet Biomedical, Madison, WI). After the electrophysiological assessment, the grip strength was measured in a standing position. 2.3. Study of post-tetanic potentiation After the baseline recording, tetanic stimulation was delivered to the median nerve at a frequency of 10 Hz (single 0.2 ms pulses delivered once every 0.1 s) for 10 s (stimulus number: 100). We confirmed in preliminary experiments that the pain during tetanic stimulation was tolerable. Using the methods described above, CMAP and MRP were recorded successively at 5, 10, 30, 60, 90 and 120 s after the tetanic stimulation (Fig. 1). These procedures were performed bilaterally in all subjects. 2.4. Statistical analysis We were not able to confirm Gaussian distribution of every group because some groups consisted of small number of subjects. Therefore, although F values were calculated by Levene test before p values, Mann–Whitney U-test was used to analyze the differences between normal and patient groups. Kruskal–Wallis test was used to compare the differences among three groups, and when a significant difference was detected, Steel–Dwass test was used to compare the differences between two groups. ANOVA fol-
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lowed by Dunnett’s test was used to analyze the changes in CMAP, MRP and excitation–contraction coupling time from baseline at various times after tetanic stimulation. A p value less than 0.05 was considered statistically significant. The JMP statistical program (SAS Institute Inc., Cary, NC) was used for data analysis. 3. Results 3.1. Clinical features Eleven MG patients comprised 2 patients with a purely ocular form (MGFA class 1) and 9 patients with generalized form (MGFA class 2–4). The mean grip strength was 19.2 kg in MG patients (11 subjects, 22 hands), 32.4 kg in healthy controls (25 subjects, 50 hands), and 16.4 kg in patients with myopathy (9 subjects, 18 hands). Mann–Whitney U-test showed significantly lower grip strength in MG and myopathy groups (p < 0.01) compared to healthy controls (Table 1). Serum antibodies against AChR were detected in 8 MG patients, and MuSK antibodies in none of the patients. Extended thymectomy was performed in 8 MG patients. Pathology demonstrated thymoma in 4 patients, remnant thymus in 2 patients, and thymic hyperplasia in 2 patients. 3.2. Electrophysiological findings at baseline For the combined data of left and right hands, the mean baseline amplitude and area of CMAP were respectively 10.6 mV and 30.0 msmV in healthy controls (25 subjects, 50 hands), 8.0 mV and 22.2 msmV in MG patients (11 subjects, 22 hands), and 9.1 mV and 25.9 msmV in patients with myopathy (9 subjects, 18 hands). These CMAP parameters were significantly lower in MG patients compared to healthy controls (p < 0.01) (Table 1). From the MRP recordings, the mean maximal acceleration of muscle twitch was 5.5 m/s2 in healthy controls, 2.9 m/s2 in MG patients and 3.2 m/s2 in patients with myopathy. The maximal acceleration was significantly lower in MG patients (p < 0.01) and in patients with myopathy (p < 0.05) compared to healthy control subjects (Table 1). 3.3. Chronological changes of electrophysiological findings after tetanic stimulation 3.3.1. Compound muscle action potential The tetanic stimulation was well tolerated, and no complication occurred during or after the study in any subject. ANOVA showed no significant changes in CMAP amplitude at 5, 10, 30, 60, 90, 120 s after tetanic stimulation in healthy control subjects (F = 0.32, p = 0.924), MG patients (F = 0.09, p = 0.997), and patients with myopathy (F = 0.04, p = 0.999) (data not shown).
Fig. 1. Post-tetanic potentiation of muscle twitch in a healthy control subject. The compound muscle action potentials (CMAPs; upper traces) of abductor pollicis brevis muscle and the movement-related potentials (MRP; lower traces) of the thumb are simultaneously recorded at baseline (A) and after tetanic stimulation at 10 Hz for 10 s (B). Note the potentiation of MRP with unchanged CMAPs after tetanic stimulation.
3.3.2. Maximal acceleration of muscle twitch In healthy control subjects, maximal acceleration of MRP (muscle twitch) changed significantly after tetanic stimulation (F = 2.88, p = 0.009). Maximal acceleration (mean ± SD) increased significantly from 5.5 ± 3.8 m/s2 at baseline to 9.5 ± 7.2 m/s2 at 5 s (p = 0.007) and 9.5 ± 7.6 m/s2 at 10 s (p = 0.007) after tetanic stimulation, and thereafter recovered to baseline level (Fig. 2C). In MG patients, maximal acceleration changed significantly (F = 2.93, p = 0.01), and increased significantly from 2.9 ± 1.0 m/s2 at baseline to 4.2 ± 1.7 m/s2 at 5 s (p = 0.009) and 4.2 ± 1.6 m/s2 at 10 s (p = 0.01), followed by recovery to baseline level (Fig. 2A). In patients with myopathy, maximal acceleration also changed significantly (F = 2.47, p = 0.03), increasing significantly from 3.2 ± 1.6 m/s2 at baseline to 5.3 ± 2.7 m/s2 at 5 s (p = 0.04) and
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5.3 ± 2.8 m/s2 at 10 s (p = 0.04), and thereafter recovering to baseline level (Fig. 2B). 3.4. Post-tetanic potentiation of muscle twitch The Kruskal–Wallis test showed significant differences in maximal acceleration of MRP among three groups, both at baseline (p = 0.0007) and at 5 s after tetanic stimulation (p < 0.0001) (Fig. 3), but no significant difference at 10 s after tetanic stimulation (p = 0.0595; data not shown). The Steel–Dwass test showed significantly lower accelerations in MG and myopathy patients compared to healthy controls at baseline (p = 0.002 and 0.03, respectively; Fig. 3A) and at 5 s after tetanic stimulation (p < 0.0001 and p = 0.04, respectively; Fig. 3B). Maximal acceleration was not significantly different between MG and myopathy patients both before and 5 s after tetanic stimulation (Fig. 3A,B). However, the ratio of acceleration (5 s after tetanic stimulation/ baseline), which indicates post-tetanic potentiation, was significantly lower in MG patients compared to healthy controls (p = 0.01) but was not significantly different between myopathy patients and healthy controls (Fig. 3C). 4. Discussion
Fig. 2. Increase in maximal acceleration after tetanic stimulation in patients with myasthenia gravis (A) and myopathy (B), and healthy controls (C). Closed circles denote raw data of the maximal acceleration in each group. Open circles and vertical bars indicate mean and standard deviation at each time. The connecting lines indicate the chronological changes of mean values. ANOVA followed by Dunnett’s test detected a significant increase in maximal acceleration from baseline, at 5 and 10 s after tetanic stimulation in three groups.
In the present method, the amplitude and area of CMAP did not change significantly after tetanic stimulation compared to baseline. In our study, we verified carefully that the muscle lengths did not change after tetanic stimulation. In addition, the unchanged CMAP parameters confirm that muscle lengths were the same during all recordings even after tetanic stimulation. If muscle shortening remains after tetanic stimulation, the movement-induced effect would increase the amplitude and decrease the duration of CMAP (Hashimoto et al., 1994). The stable CMAP also implies that the post-tetanic potentiation may not be derived from accumulation of calcium in the nerve axon or mobilization of ACh vesicles in the presynaptic terminals (Maddison et al., 1998). The excitation–contraction coupling in skeletal muscle includes a number of steps. After generation of a muscle action potential, sarcolemmal depolarization spreads transversely to the T tubules. The voltage sensor of the T tubules transmits the electric charge to the ryanodine receptor, resulting in channel opening. This opening leads to Ca2+ release from the sarcoplasmic reticulum into the myoplasm. Binding of the released Ca2+ to troponin triggers an interaction between actin filaments and myosin filaments. Finally, these filaments slide and muscle fiber length shortens (Beam and Horowicz, 2004). Although the step and site involved in dysfunction of the process have not been precisely identified, the present
Fig. 3. Comparisons between three groups for maximal acceleration at baseline (A), maximal acceleration at 5 s after tetanic stimulation (B), and the ratio of maximal acceleration (5 s after tetanic stimulation divided by baseline) (C). Closed circles denote raw data of each parameter. Open circles and vertical bars indicate mean and standard deviation. The Steel–Dwass test detected a significant decrease in the ratio only in MG compared to healthy controls (C).
D. Yamamoto et al. / Clinical Neurophysiology 127 (2016) 1689–1693
study strongly supports our previous hypothesis that impairment of excitation–contraction coupling contributes to muscle weakness in MG (Tsuda et al., 2010; Imai et al., 2011, 2012a,b). Tetanic stimulation is known to be useful to elucidate the time course of the mechanical force of contraction. The force of muscle twitch increases during prolonged stimulation in healthy control subjects, but not in patients with MG. This phenomenon, called a positive staircase, has provided scientific significance, but not diagnostic specificity as a clinical test (Desmedt et al., 1973). Our results indicate that muscle twitch at baseline as well as that after tetanic stimulation was impaired in MG and myopathy, while there was no significant difference in maximal acceleration of muscle twitch between MG and myopathy. However, the ratio of acceleration (5 s after tetanic stimulation/baseline), which indicates posttetanic potentiation, was significantly diminished only in MG compared to healthy control subjects (Steel–Dwass test p = 0.01) and was not significantly different between MG and myopathy. These results indicate that our methods may have relatively high sensitivity to detect impaired post-tetanic potentiation of muscle twitch in MG, but relatively low specificity in discriminating MG from myopathy. Our method is potentially useful as a clinical testing procedure because it is easy to measure in a relatively short time compared to previous methods using a strain gauge (Krarup, 1977; Krarup and Horowitz, 1979; Slomic´ et al., 1968), and is well tolerated without any adverse effects (Kimura, 2013). Thus our method is novel in the sense that post-tetanic potentiation in MG is assessed using a compact accelerometer instead of a largescale equipment with strain gauge. At this stage, this technique may be useful for research purpose, such as in the elucidation of the relationship between excitation–contraction coupling impairment and myasthenic features in MG. Further study is warranted to examine the feasibility of clinical application of this method. Acknowledgements This study was supported in part by Grant-in Aid for Scientific Research (No. 25461321, Tomihiro Imai) from Japan Society for the Promotion of Science. We wish to thank Dr. Masakatsu Motomura (Medical Engineering Course, The Department of Engineering, The Faculty of Engineering, Nagasaki Institute of Applied Science) and Dr. Kiyoe Ohta (Clinical Research Center, National Hospital Organization Utano Hospital) for assaying anti-MuSK antibodies. Conflict of interest: There are no competing interests. References Beam KG, Horowicz P. Excitation–contraction coupling in skeletal muscle. 3rd ed. In: Engel AG, Clara Franzini-Armstrong L, editors. Myology. New York: McGrawHill; 2004. p. 257–80.
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Bowditch HP. Über die Eigenthümlichkeinen der Reizbarkeit, welche die Muskelfasern des Herzens zeigen. Arb Physiol Anst (Leipzig) 1870/71;6:139–76. Brown GL, von Euler US. The after-effects of a tetanus on mammalian muscle. J Physiol 1938;93:39–60. Desmedt JE, Emeryk B, Hainaut K, Reinhold H, Borenstein S. Muscular dystrophy and myasthenia gravis. Muscle contraction properties studied by the staircase phenomenon. In: Desmedt JE, editor. New developments in electromyography and clinical neurophysiology 1973;vol. 1. Basel: Karger; 1973. p. 380–99. Hashimoto S, Kawamura J, Segawa Y, Harada Y, Hanakawa T, Osaki Y. Waveform changes of compound muscle action potential (CMAP) with muscle length. J Neurol Sci 1994;124:21–4. Imai T, Tsuda E, Toyoshima T, Yoshikawa H, Motomura M, Shimohama S. Anti-ryanodine receptor–positive acetylcholine receptor–negative myasthenia gravis: evidence of impaired E–C coupling. Muscle Nerve 2011;43:294–5. Imai T, Tsuda E, Hozuki T, Yoshikawa H, Yamauchi R, Saitoh M, et al. Contribution of anti-ryanodine receptor antibody to impairment of excitation–contraction coupling in myasthenia gravis. Clin Neurophysiol 2012a;123:1242–7. Imai T, Tsuda E, Hozuki T, Yamauchi R, Saitoh M, Hisahara S, et al. Early effect of tacrolimus in improving excitation–contraction coupling in myasthenia gravis. Clin Neurophysiol 2012b;123:1886–90. Jaretzki A, Barohn RJ, Ernstoff RM, Kaminski HJ, Keesey JC, Penn AS, et al. Myasthenia gravis: recommendations for clinical research standards. Neurology 2000;55:16–23. Kimura J. Electrodiagnosis in diseases of nerve and muscle: principles and practice. 4th ed. New York: Oxford University Press; 2013. p. 449–74. Kokubun N, Sonoo M, Imai T, Arimura Y, Kuwabara S, Komori T, et alThe Japanese SFEMG Study Group. Reference values for voluntary and stimulated single-fiber EMG using concentric needle electrodes: a multi-center prospective study. Clin Neurophysiol 2012;123:613–20. Kouyoumdjian JA, Stålberg E. Reference jutter values for concentric needle electrodes in voluntarily activated extensor digitorum communis and orbicularis oculi muscles. Muscle Nerve 2008;37:694–9. Krarup C. Electrical and mechanical responses in the platysma and in the adductor pollicis muscle: in patients with myasthenia gravis. J Neurol Neurosurg Psychiatry 1977;40:241–9. Krarup C, Horowitz SH. Evoked responses of the elbow flexors in control subjects and in myopathy patients. Muscle Nerve 1979;2:465–77. Maddison P, Newsom-Davis J, Mills KR. Decay of postexercise augmentation in the Lambert-Eaton myasthenic syndrome. Effect of cooling. Neurology 1998;50:1083–7. Nakata M, Kuwabara S, Kawaguchi N, Takahashi H, Misawa S, Kanai K, et al. Is excitation–contraction coupling impaired in myasthenia gravis? Clin Neurophysiol 2007;118:1144–8. Pagala MKD, Nandakumar NV, Venkatachari SAT, Ravindran K, Namba T, Grob D. Responses of intercostals muscle biopsies from normal subjects and patients with myasthenia gravis. Muscle Nerve 1990;13:1012–22. Pagala MKD, Nandakumar NV, Venkatachari SAT, Ravindran K, Amaladevi B, Namba T, et al. Mechanisms of fatigue in normal intercostal muscle and muscle from patients with myasthenia gravis. Muscle Nerve 1993;16:911–21. Shiraishi H, Motomura M, Yoshimura T, Fukudome T, Fukuda T, Nakao Y, et al. Acetylcholine receptors loss and postsynaptic damage in MuSK antibodypositive myasthenia gravis. Ann Neurol 2005;57:289–93. Slomic´ A, Rosenfalck A, Buchthal F. Electrical and mechanical responses of normal and myasthenic muscle: with particular reference to the staircase phenomenon. Brain Res 1968;10:1–78. Tsuda E, Imai T, Hozuki T, Yamauchi R, Saitoh M, Hisahara S, et al. Correlation of bite force with excitation–contraction coupling time of the masseter in myasthenia gravis. Clin Neurophysiol 2010;121:1051–8.
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Clinical Neurophysiology journal homepage: www.elsevier.com/locate/clinph
Impaired post-tetanic potentiation of muscle twitch in myasthenia gravis Daisuke Yamamoto a, Tomihiro Imai b,⇑, Emiko Tsuda a, Takayoshi Hozuki c, Rika Yamauchi d, Shin Hisahara a, Jun Kawamata a, Shun Shimohama a a
Department of Neurology, Sapporo Medical University School of Medicine, Sapporo, Japan Department of Occupational Therapy, Sapporo Medical University School of Health Sciences, Sapporo, Japan Sapporo Shirakabadai Hospital, Sapporo, Japan d Department of Neurology, Sunagawa City Medical Center, Sunagawa, Japan b c
a r t i c l e
i n f o
Article history: Accepted 10 October 2015 Available online 23 October 2015 Keywords: Myasthenia gravis Muscle fatigue Staircase phenomenon Excitation–contraction coupling Post-tetanic potentiation
h i g h l i g h t s A novel method using an accelerometer was used to evaluate post-tetanic potentiation of muscle
twitch in myasthenia gravis. Significant decrease in post-tetanic potentiation of muscle twitch may be associated with impairment
of excitation–contraction coupling in myasthenia gravis. The present method has high sensitivity in detecting impairment of excitation–contraction coupling
in myasthenia gravis.
a b s t r a c t Objective: The aim of this study was to evaluate post-tetanic potentiation of muscle twitch in myasthenia gravis (MG). Methods: Post-tetanic potentiation was evaluated by recording the compound muscle action potential (CMAP) of abductor pollicis brevis and movement-related potential (MRP) of the thumb using an accelerometer after tetanic stimulation of the median nerve at the wrist. After baseline recording, tetanic stimulation was delivered to the median nerve at a frequency of 10 Hz for 10 s. The CMAP and MRP were successively recorded at baseline and at 5, 10, 30, 60, 90 and 120 s after tetanic stimulation. The chronological changes of CMAPs and MRPs were recorded bilaterally in 11 patients with MG, 9 patients with myopathies (disease controls), and 25 healthy control subjects. Results: Maximal acceleration of MRP was significantly elevated during 10 s after tetanic stimulation without any CMAP changes in all groups. However, statistical analysis detected a significant decrease in post-tetanic potentiation of maximal acceleration of MRP in MG patients only compared to healthy controls, but not in myopathy patients, which may imply impairment of excitation–contraction coupling in MG. Conclusions: Post-tetanic potentiation of muscle twitch is significantly diminished in MG, suggesting impaired excitation–contraction coupling. Significance: Measurement of post-tetanic potentiation using an accelerometer is a simple and sensitive method to detect impairment of excitation–contraction coupling in MG. Ó 2015 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction
⇑ Corresponding author at: Department of Occupational Therapy, Sapporo Medical University School of Health Sciences, South 1, West 17, Chuo-ku, Sapporo 060-8556, Japan. Tel.: +81 11 611 2111; fax: +81 11 611 2115. E-mail address: [email protected] (T. Imai).
Although defective neuromuscular transmission is known to be a cause of muscle weakness in myasthenia gravis (MG), previous studies have shown the possible roles of other processes, the failure of which may also cause muscle weakness in MG. Especially,
http://dx.doi.org/10.1016/j.clinph.2015.10.037 1388-2457/Ó 2015 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.
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excitation–contraction coupling may be defective in patients with MG (Pagala et al., 1990, 1993; Slomic´ et al., 1968). The excitation– contraction coupling in skeletal muscle is the process whereby an action potential triggers a muscle fiber to contract. Some investigators have applied the post-tetanic potentiation and/or the staircase phenomenon to elucidate the impairment of excitation–contraction coupling in MG (Krarup, 1977; Slomic´ et al., 1968). Posttetanic potentiation is the enhancement of active twitch force following high-frequency tetanic stimulation (Brown and von Euler, 1938), while the staircase phenomenon is the progressive increase in active twitch force during repetitive low-frequency stimulation (Bowditch, 1870/71). The post-tetanic potentiation and staircase phenomenon have been explained by an increase in sarcoplasmic Ca2+, which may induce facilitation of excitation–contraction coupling after tetanic stimulation and stepwise increase in response during repetitive twitch, respectively. Studies in MG patients have demonstrated a correlation between the magnitude of potentiation and the severity of MG, and proposed the significance of impaired excitation–contraction coupling in MG (Krarup, 1977; Slomic´ et al., 1968). However, post-tetanic potentiation and staircase phenomenon are rarely used to evaluate the impairment of excitation–contraction coupling in the clinical laboratory, presumably because complicated apparatus using a strain gauge is required. In this study, we describe a novel procedure to measure posttetanic potentiation using an accelerometer attached to the hand muscle instead of the complicated apparatus with a strain gauge used in previous studies. Our method is simple and may be a useful clinical test for the detection of impairment of excitation–contraction coupling in MG.
2. Materials and methods 2.1. Subjects and protocol We studied 11 patients with MG (3 males and 8 females; aged 33–67 years, mean 47.2 years) at Sapporo Medical University
Hospital. The diagnosis of MG was based on typical clinical features and electrophysiological evidence of a defect in neuromuscular transmission, which is either an abnormal decrement in repetitive nerve stimulation tests (muscles tested: orbicularis oculi, nasalis, trapezius, abductor pollicis brevis and abductor digiti minimi), or increased jitter on concentric needle single fiber electromyography of the voluntarily activated extensor digitorum communis and orbicularis oculi muscles (Kokubun et al., 2012; Kouyoumdjian and Stålberg, 2008). Acetylcholine receptor (AChR) binding antibody was measured in a commercial laboratory using a radioimmunoassay. In MG patients without AChR antibody, serum antibody against muscle-specific receptor tyrosine kinase (MuSK) was measured by immunoprecipitation of 125I-recombinant MuSK extracellular domains (Shiraishi et al., 2005). The diagnosis of thymic lesion was based on histopathological findings after extended thymectomy. At the time of the present study, 1 of 11 patients had never received any specific MG therapy, and the remaining 10 patients showed exacerbation of myasthenic symptoms despite receiving corticosteroids alone (5 patients), or corticosteroids combined with tacrolimus (4 patients) or cyclosporine (1 patient). Extended thymectomy had already been performed in 8 patients. Disease severity was graded according to the clinical classification of Myasthenia Gravis Foundation of America (MGFA) (Jaretzki et al., 2000). We also studied 25 healthy control subjects (9 males and 16 females) aged 26–60 years (mean, 40.2 years) to establish the normal ranges for the methods used in this study, and 9 patients with myopathies (3 males and 6 females) aged 21–66 years (mean, 48.4 years) to examine whether myopathic changes influence excitation–contraction coupling (Table 1). Myopathies included autoimmune myositis, muscular dystrophy and muscle sarcoidosis. All the healthy control subjects and patients gave informed consent for participation in this study. The study was approved by the Ethics Committee, Sapporo Medical University Hospital, Sapporo, Japan.
Table 1 Comparison of age, grip power and electrophysiological findings in subject groups. Subject group
Number of subjects (males, females) Age (years) Number of hands tested
F value
Healthy control
Myasthenia gravis
Myopathy
25 (9, 16) 40.2 (11.3, 27–60) 50
11 (2, 9) 47.2 (12.8, 33–67) 22
9 (3, 6) 48.4 (14.0, 21–66) 18
Grip power (kg)
Lt + Rt Lt Rt
32.4 (8.5, 21–54) 31.0 (7.5, 21–46) 33.7 (9.4, 23–54)
19.2 (10.3, 5–48)⁄⁄ 18.9 (9.6, 10–43)⁄⁄ 19.5 (11.3, 5–48)⁄⁄
16.4 (8.8, 3.5–35)⁄⁄ 15.4 (9.4, 3.5–32)⁄⁄ 17.4 (8.7, 5–35)⁄⁄
0.09 0.25 0.38
CMAP amplitude (mV)
Lt + Rt Lt Rt
10.6 (1.9, 6.9–13.8) 11.0 (1.7, 7.0–13.5) 10.3 (2.0, 6.9–13.8)
8.0 (2.7, 3.5–14.3)⁄⁄ 7.9 (2.5, 4.1–11.2)⁄⁄ 8.2 (3.0, 3.5–14.3)
9.1 (3.4, 1.5–15.1) 9.3 (3.3, 3.1–13.3) 8.8 (3.8, 1.5–15.1)
4.60 3.00 2.16
CMAP area (msmV)
Lt + Rt Lt Rt
30.0 (5.7, 17.7–42.6) 30.5 (5.2, 22.5–42.6) 29.4 (6.2, 17.7–42.1)
22.2 (7.9, 10.2–39.8)⁄⁄ 22.9 (9.1, 10.3–39.8)⁄ 21.5 (7.0, 10.2–35.3)⁄⁄
25.9 (10.5, 4.0–42.4) 26.5 (11.0, 6.8–42.4) 25.3 (10.7, 4.0–40.2)
5.12 4.67 1.32
Maximal acceleration at baseline (m/s2)
Lt + Rt Lt Rt
5.5 (3.8, 1.6–17.6) 5.6 (4.0, 1.8–17.6) 5.4 (3.6, 1.6–14.7)
2.9 (1.0, 0.8–5.3)⁄⁄ 2.9 (0.9, 1.6–4.0)⁄⁄ 2.8 (1.2, 0.8–5.3)
3.2 (1.6, 0.6–7.4)⁄ 3.2 (1.4, 0.6–5.1) 3.2 (1.8, 1.2–7.4)
8.64 3.26 6.06
Maximal acceleration at 5 s after TS (m/s2)
Lt + Rt Lt Rt
9.5 (7.2, 2.7–36.0) 9.9 (7.9, 3.1–36.0) 9.0 (6.6, 2.7–28.1)
4.2 (1.7, 1.1–8.1)⁄⁄ 4.3 (1.6, 2.1–6.5)⁄⁄ 4.0 (1.8, 1.1–8.1)⁄
5.3 (2.7, 1.0–10.3)⁄ 5.4 (2.9, 1.0–9.5) 5.2 (2.7, 1.7–10.3)
8.99 3.57 6.17
PTP of MRP acceleration (5 s after TS divided by baseline)
Lt + Rt Lt Rt
1.7 (0.4, 1.0–2.6) 1.8 (0.4, 1.2–2.6) 1.7 (0.3, 1.0–2.2)
1.5 (0.2, 1.0–2.0)⁄⁄ 1.4 (0.2, 1.2–1.9) 1.5 (0.3, 1.0–2.0)
1.7 (0.4, 1.0–2.2) 1.7 (0.4, 1.0–2.2) 1.6 (0.3, 1.1–2.1)
1.56 1.97 0.16
Data are expressed as mean (SD, range) for age, grip power, CMAP amplitude, CMAP area, ECCT, and acceleration parameters. Single asterisk and double asterisks denote p < 0.05 and p < 0.01, respectively, compared to healthy control values, as analyzed by Mann–Whitney U-test. CMAP, compound muscle action potential; MRP, movementrelated potential; ECCT, excitation–contraction coupling time; TS, tetanic stimulation; PTP, post-tetanic potentiation, Lt, left hand; Rt, right hand.
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2.2. Measurement of baseline compound muscle action potentials and movement-related potentials Compound muscle action potentials (CMAPs) were recorded from the abductor pollicis brevis using surface disc electrodes in belly-tendon arrangement after median nerve stimulation at the wrist. Simultaneously, movement-related potentials (MRPs) were recorded using an accelerometer (SV1101, NEC, Japan) taped to the tip of the thumb (Nakata et al., 2007). The amplitude and area of CMAP were measured from the baseline to the negative peak using a cursor. The maximal acceleration of thumb movement was obtained from the initial peak amplitude of MRP. For stimulation, a 0.2-ms rectangular pulse was delivered to the median nerve at the wrist with gradually increasing intensity to reach a supramaximal response. Once a supramaximal CMAP was obtained, the initial recording of resting CMAP and MRP was performed (baseline). All stimulating and recording procedures were performed using an electromyograph (Nicolet Viking IV, Nicolet Biomedical, Madison, WI). After the electrophysiological assessment, the grip strength was measured in a standing position. 2.3. Study of post-tetanic potentiation After the baseline recording, tetanic stimulation was delivered to the median nerve at a frequency of 10 Hz (single 0.2 ms pulses delivered once every 0.1 s) for 10 s (stimulus number: 100). We confirmed in preliminary experiments that the pain during tetanic stimulation was tolerable. Using the methods described above, CMAP and MRP were recorded successively at 5, 10, 30, 60, 90 and 120 s after the tetanic stimulation (Fig. 1). These procedures were performed bilaterally in all subjects. 2.4. Statistical analysis We were not able to confirm Gaussian distribution of every group because some groups consisted of small number of subjects. Therefore, although F values were calculated by Levene test before p values, Mann–Whitney U-test was used to analyze the differences between normal and patient groups. Kruskal–Wallis test was used to compare the differences among three groups, and when a significant difference was detected, Steel–Dwass test was used to compare the differences between two groups. ANOVA fol-
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lowed by Dunnett’s test was used to analyze the changes in CMAP, MRP and excitation–contraction coupling time from baseline at various times after tetanic stimulation. A p value less than 0.05 was considered statistically significant. The JMP statistical program (SAS Institute Inc., Cary, NC) was used for data analysis. 3. Results 3.1. Clinical features Eleven MG patients comprised 2 patients with a purely ocular form (MGFA class 1) and 9 patients with generalized form (MGFA class 2–4). The mean grip strength was 19.2 kg in MG patients (11 subjects, 22 hands), 32.4 kg in healthy controls (25 subjects, 50 hands), and 16.4 kg in patients with myopathy (9 subjects, 18 hands). Mann–Whitney U-test showed significantly lower grip strength in MG and myopathy groups (p < 0.01) compared to healthy controls (Table 1). Serum antibodies against AChR were detected in 8 MG patients, and MuSK antibodies in none of the patients. Extended thymectomy was performed in 8 MG patients. Pathology demonstrated thymoma in 4 patients, remnant thymus in 2 patients, and thymic hyperplasia in 2 patients. 3.2. Electrophysiological findings at baseline For the combined data of left and right hands, the mean baseline amplitude and area of CMAP were respectively 10.6 mV and 30.0 msmV in healthy controls (25 subjects, 50 hands), 8.0 mV and 22.2 msmV in MG patients (11 subjects, 22 hands), and 9.1 mV and 25.9 msmV in patients with myopathy (9 subjects, 18 hands). These CMAP parameters were significantly lower in MG patients compared to healthy controls (p < 0.01) (Table 1). From the MRP recordings, the mean maximal acceleration of muscle twitch was 5.5 m/s2 in healthy controls, 2.9 m/s2 in MG patients and 3.2 m/s2 in patients with myopathy. The maximal acceleration was significantly lower in MG patients (p < 0.01) and in patients with myopathy (p < 0.05) compared to healthy control subjects (Table 1). 3.3. Chronological changes of electrophysiological findings after tetanic stimulation 3.3.1. Compound muscle action potential The tetanic stimulation was well tolerated, and no complication occurred during or after the study in any subject. ANOVA showed no significant changes in CMAP amplitude at 5, 10, 30, 60, 90, 120 s after tetanic stimulation in healthy control subjects (F = 0.32, p = 0.924), MG patients (F = 0.09, p = 0.997), and patients with myopathy (F = 0.04, p = 0.999) (data not shown).
Fig. 1. Post-tetanic potentiation of muscle twitch in a healthy control subject. The compound muscle action potentials (CMAPs; upper traces) of abductor pollicis brevis muscle and the movement-related potentials (MRP; lower traces) of the thumb are simultaneously recorded at baseline (A) and after tetanic stimulation at 10 Hz for 10 s (B). Note the potentiation of MRP with unchanged CMAPs after tetanic stimulation.
3.3.2. Maximal acceleration of muscle twitch In healthy control subjects, maximal acceleration of MRP (muscle twitch) changed significantly after tetanic stimulation (F = 2.88, p = 0.009). Maximal acceleration (mean ± SD) increased significantly from 5.5 ± 3.8 m/s2 at baseline to 9.5 ± 7.2 m/s2 at 5 s (p = 0.007) and 9.5 ± 7.6 m/s2 at 10 s (p = 0.007) after tetanic stimulation, and thereafter recovered to baseline level (Fig. 2C). In MG patients, maximal acceleration changed significantly (F = 2.93, p = 0.01), and increased significantly from 2.9 ± 1.0 m/s2 at baseline to 4.2 ± 1.7 m/s2 at 5 s (p = 0.009) and 4.2 ± 1.6 m/s2 at 10 s (p = 0.01), followed by recovery to baseline level (Fig. 2A). In patients with myopathy, maximal acceleration also changed significantly (F = 2.47, p = 0.03), increasing significantly from 3.2 ± 1.6 m/s2 at baseline to 5.3 ± 2.7 m/s2 at 5 s (p = 0.04) and
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5.3 ± 2.8 m/s2 at 10 s (p = 0.04), and thereafter recovering to baseline level (Fig. 2B). 3.4. Post-tetanic potentiation of muscle twitch The Kruskal–Wallis test showed significant differences in maximal acceleration of MRP among three groups, both at baseline (p = 0.0007) and at 5 s after tetanic stimulation (p < 0.0001) (Fig. 3), but no significant difference at 10 s after tetanic stimulation (p = 0.0595; data not shown). The Steel–Dwass test showed significantly lower accelerations in MG and myopathy patients compared to healthy controls at baseline (p = 0.002 and 0.03, respectively; Fig. 3A) and at 5 s after tetanic stimulation (p < 0.0001 and p = 0.04, respectively; Fig. 3B). Maximal acceleration was not significantly different between MG and myopathy patients both before and 5 s after tetanic stimulation (Fig. 3A,B). However, the ratio of acceleration (5 s after tetanic stimulation/ baseline), which indicates post-tetanic potentiation, was significantly lower in MG patients compared to healthy controls (p = 0.01) but was not significantly different between myopathy patients and healthy controls (Fig. 3C). 4. Discussion
Fig. 2. Increase in maximal acceleration after tetanic stimulation in patients with myasthenia gravis (A) and myopathy (B), and healthy controls (C). Closed circles denote raw data of the maximal acceleration in each group. Open circles and vertical bars indicate mean and standard deviation at each time. The connecting lines indicate the chronological changes of mean values. ANOVA followed by Dunnett’s test detected a significant increase in maximal acceleration from baseline, at 5 and 10 s after tetanic stimulation in three groups.
In the present method, the amplitude and area of CMAP did not change significantly after tetanic stimulation compared to baseline. In our study, we verified carefully that the muscle lengths did not change after tetanic stimulation. In addition, the unchanged CMAP parameters confirm that muscle lengths were the same during all recordings even after tetanic stimulation. If muscle shortening remains after tetanic stimulation, the movement-induced effect would increase the amplitude and decrease the duration of CMAP (Hashimoto et al., 1994). The stable CMAP also implies that the post-tetanic potentiation may not be derived from accumulation of calcium in the nerve axon or mobilization of ACh vesicles in the presynaptic terminals (Maddison et al., 1998). The excitation–contraction coupling in skeletal muscle includes a number of steps. After generation of a muscle action potential, sarcolemmal depolarization spreads transversely to the T tubules. The voltage sensor of the T tubules transmits the electric charge to the ryanodine receptor, resulting in channel opening. This opening leads to Ca2+ release from the sarcoplasmic reticulum into the myoplasm. Binding of the released Ca2+ to troponin triggers an interaction between actin filaments and myosin filaments. Finally, these filaments slide and muscle fiber length shortens (Beam and Horowicz, 2004). Although the step and site involved in dysfunction of the process have not been precisely identified, the present
Fig. 3. Comparisons between three groups for maximal acceleration at baseline (A), maximal acceleration at 5 s after tetanic stimulation (B), and the ratio of maximal acceleration (5 s after tetanic stimulation divided by baseline) (C). Closed circles denote raw data of each parameter. Open circles and vertical bars indicate mean and standard deviation. The Steel–Dwass test detected a significant decrease in the ratio only in MG compared to healthy controls (C).
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study strongly supports our previous hypothesis that impairment of excitation–contraction coupling contributes to muscle weakness in MG (Tsuda et al., 2010; Imai et al., 2011, 2012a,b). Tetanic stimulation is known to be useful to elucidate the time course of the mechanical force of contraction. The force of muscle twitch increases during prolonged stimulation in healthy control subjects, but not in patients with MG. This phenomenon, called a positive staircase, has provided scientific significance, but not diagnostic specificity as a clinical test (Desmedt et al., 1973). Our results indicate that muscle twitch at baseline as well as that after tetanic stimulation was impaired in MG and myopathy, while there was no significant difference in maximal acceleration of muscle twitch between MG and myopathy. However, the ratio of acceleration (5 s after tetanic stimulation/baseline), which indicates posttetanic potentiation, was significantly diminished only in MG compared to healthy control subjects (Steel–Dwass test p = 0.01) and was not significantly different between MG and myopathy. These results indicate that our methods may have relatively high sensitivity to detect impaired post-tetanic potentiation of muscle twitch in MG, but relatively low specificity in discriminating MG from myopathy. Our method is potentially useful as a clinical testing procedure because it is easy to measure in a relatively short time compared to previous methods using a strain gauge (Krarup, 1977; Krarup and Horowitz, 1979; Slomic´ et al., 1968), and is well tolerated without any adverse effects (Kimura, 2013). Thus our method is novel in the sense that post-tetanic potentiation in MG is assessed using a compact accelerometer instead of a largescale equipment with strain gauge. At this stage, this technique may be useful for research purpose, such as in the elucidation of the relationship between excitation–contraction coupling impairment and myasthenic features in MG. Further study is warranted to examine the feasibility of clinical application of this method. Acknowledgements This study was supported in part by Grant-in Aid for Scientific Research (No. 25461321, Tomihiro Imai) from Japan Society for the Promotion of Science. We wish to thank Dr. Masakatsu Motomura (Medical Engineering Course, The Department of Engineering, The Faculty of Engineering, Nagasaki Institute of Applied Science) and Dr. Kiyoe Ohta (Clinical Research Center, National Hospital Organization Utano Hospital) for assaying anti-MuSK antibodies. Conflict of interest: There are no competing interests. References Beam KG, Horowicz P. Excitation–contraction coupling in skeletal muscle. 3rd ed. In: Engel AG, Clara Franzini-Armstrong L, editors. Myology. New York: McGrawHill; 2004. p. 257–80.
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