Muscle Velocity Recovery Cycles to Examine Muscle Membrane Properties


Your institution must subscribe to JoVE's Neuroscience section to access this content.

Fill out the form below to receive a free trial or learn more about access:



Presented here is a protocol for the recording of muscle velocity recovery cycles (MVRCs), a new method of examining muscle membrane properties. MVRCs enable in vivo assessment of muscle membrane potential and alterations in muscle ion channel function in relation to pathology, and it enables the demonstration of muscle depolarization in neurogenic muscles.

Cite this Article

Copy Citation | Download Citations | Reprints and Permissions

Witt, A., Bostock, H., Z'Graggen, W. J., Tan, S. V., Kristensen, A. G., Kristensen, R. S., Larsen, L. H., Zeppelin, Z., Tankisi, H. Muscle Velocity Recovery Cycles to Examine Muscle Membrane Properties. J. Vis. Exp. (156), e60788, doi:10.3791/60788 (2020).


Although conventional nerve conduction studies (NCS) and electromyography (EMG) are suitable for the diagnosis of neuromuscular disorders, they provide limited information about muscle fiber membrane properties and underlying disease mechanisms. Muscle velocity recovery cycles (MVRCs) illustrate how the velocity of a muscle action potential depends on the time after a preceding action potential. MVRCs are closely related to changes in membrane potential that follow an action potential, thereby providing information about muscle fiber membrane properties. MVRCs may be recorded quickly and easily by direct stimulation and recording from multi-fiber bundles in vivo. MVRCs have been helpful in understanding disease mechanisms in several neuromuscular disorders. Studies in patients with channelopathies have demonstrated the different effects of specific ion channel mutations on muscle excitability. MVRCs have been previously tested in patients with neurogenic muscles. In this prior study, muscle relative refraction period (MRRP) was prolonged, and early supernormality (ESN) and late supernormality (LSN) were reduced in patients compared to healthy controls. Thereby, MVRCs can provide in vivo evidence of membrane depolarization in intact human muscle fibers that underlie their reduced excitability. The protocol presented here describes how to record MVRCs and analyze the recordings. MVRCs can serve as a fast, simple, and useful method for revealing disease mechanisms across a broad range of neuromuscular disorders.


Nerve conduction studies (NCS) and electromyography (EMG) are the conventional electrophysiological methods used for the diagnosis of neuromuscular disorders. NCS enables detection of axonal loss and demyelination in the nerves1, while EMG can differentiate whether myopathy or neurogenic changes are present in the muscle due to nerve damage. However, NCS or EMG provide limited information about muscle fiber membrane properties and underlying disease mechanisms. This information can be achieved using intracellular electrodes in isolated muscles from muscle biopsies2,3,4. However, it is of clinical importance to use methodologies using recordings from intact muscles in patients.

The velocity of a second muscle fiber action potential changes as a function of the delay after the first5, and this velocity recovery function (or recovery cycle) has been shown to change in dystrophic or denervated muscles. The yield of such recordings from single muscle fibers was, however, too low to be of use as a clinical tool6. However, Z'Graggen and Bostock later found that multi-fiber recordings, obtained by direct stimulation and recording from the same bundle of muscle fibers, provide a fast and simple method of obtaining such recordings in vivo7. A sequence of paired pulse electrical stimuli with varying interstimulus intervals (ISIs) is used in this method7,8,9,10,11.

The evaluated MVRC parameters include the following: 1) muscle relative refractory period (MRRP), which is the duration after a muscle action potential until the next action potential can be elicited; 2) early supernormality (ESN); and 3) late supernormality (LSN). ESN and LSN are the periods after the refractory period in which the action potentials are conducted along the muscle membrane faster than normal. The depolarizing afterpotential, and potassium accumulation in the t-tubules of the muscle respectively, are hypothesized as the main causes for the two periods of supernormality.

The wide applicability of MVRCs to muscle disorders has been shown in detecting membrane depolarization in ischemia7,10,12 and renal failure13, as well as providing information about muscle membrane abnormalities in critical illness myopathy14 and inclusion body myositis15. Frequency ramp and intermittent 15 Hz and 20 Hz simulation protocols have since been introduced. MVRCs, together with these additional protocols, have demonstrated the different effects on muscle membrane excitability related to loss-of-function or gain-of-function mutations in various muscle ion channels in the inherited muscle ion channelopathies (i.e., sodium channel myotonia, paramyotonia congenita16, myotonic dystrophy17, Andersen-Tawil syndrome18, and myotonia congenita19,20).

In a recent study, the applicability of MVRCs to neurogenic muscles was shown for the first time. The term "neurogenic muscle" refers to the secondary changes in skeletal muscles that develop as denervation and reinnervation after any injury to the anterior horn cells or motor axons. Denervation is characterized in EMG as spontaneous activity (i.e., fibrillations [fibs] and positive sharp waves [psws]), while large motor unit potentials with prolonged duration and increased amplitude present reinnervation21. EMG changes are evident in denervated muscles, but the underlying cellular changes in muscle fiber membrane potentials have only been demonstrated in experimental studies on isolated muscle tissue2,3,4. MVRCs provide further insight into in vivo human muscle membrane properties regarding the denervation process.

This paper describes the methodology of MVRCs in detail. It also summarizes the changes in neurogenic muscles in a subgroup of patients from a previously reported study22 and healthy control subjects that enables determination of whether the method is appropriate for a planned study.

The recordings are performing using a recording protocol that is part of a software program. Other equipment used is an isolated linear bipolar constant current stimulator, 50 Hz noise eliminator, isolated electromyography amplifier, and analogue-to-digital converter.

Subscription Required. Please recommend JoVE to your librarian.


All subjects must provide written consent prior to examination, and the protocol must be approved by the appropriate local ethical review board. All methods described here were approved by the Regional Scientific Ethical Committee and Danish Data Protection Agency.

1. Preparation of the subject

  1. Assess subjects' medical histories to ensure that they do not have any previous nervous system disorders other than the disease group that will be investigated.
  2. Inform the subject in detail about the examinations and request to obtain written consent.
    1. Inform the subject about the insertion of two needles in a leg muscle and that the muscle fibers will be stimulated with weak current.
    2. Explain that the sensation may feel slightly unpleasant.
    3. Inform the subject that the stimulation can be turned off immediately at any moment during the recording in case of any discomfort.
  3. Clean the subject's lower leg with alcohol.
  4. Insert the stimulating monopolar needle electrode (25 mm x 26 G) over the anterior tibial muscle and adhesive surface electrode as the anode 1 cm distal to the monopolar needle (Figure 1).
  5. Place a ground electrode distal to the anode.
  6. Insert the recording concentric needle electrode (25 mm x 30 G) about 2cm proximal to the stimulating monopolar needle electrode along the muscle fibers (Figure 1).
  7. Connect the recording concentric needle and ground electrodes to the preamplifier.
  8. Ask the subject to remain silent and avoid movement during the examination.
  9. Zero the output of the stimulator and connect the stimulating electrodes to the stimulator (Figure 1).
  10. Maintain the skin temperature between 32–36 °C using a warming lamp.

2. Recording of the MVRCs

  1. Start the semi-automated recording software using the muscle excitability recording protocol and turn on the stimulator. Stimulations will start at 2.5 mA with 1 Hz.
  2. Increase the stimulus intensity manually by hitting the Insert key until a response is recorded (max = 10 mA).
    1. Adjust the stimulating and recording needles if necessary, until recording an acceptable response with a stimulus intensity of less than 10 mA. The shape of the muscle action potential should be triphasic, if possible, and stable. Avoid large twitches of the whole muscle.
    2. Invert the muscle action potential by hitting the minus key (-) if the potential appears upside down.
      NOTE: A magenta horizontal line appears on the screen indicating the width of the action potential.
  3. Adjust the position and length of the magenta line by dragging the line with the mouse. The green horizontal line represents the baseline.
  4. Click OK to start recording the MVRCs.
  5. Select a stimulus response relationship from the main options.
  6. Increase stimulus intensity by hitting the Insert key to a max of 10 mA or tolerable.
  7. Click OK to start descending the stimulus response curve.
  8. Click OK when the test stimulus reaches zero.
  9. Set the stimulus intensity to level for stable latency.
  10. Click OK to return to the main menu.
  11. Select the option 1/2/5 conditioning stims for RC.
  12. Select a protocol from recovery cycle options (e.g., start quick recovery cycle [skip alternate delays]), which is the default.
    NOTE: The recording continues automatically for 34 steps with decreasing inter-stimulus intervals (ISIs).
  13. Make sure that muscle action potential is stable during the recording and that the needle has not moved. The screen changes automatically to main options when the 34 steps have completed.
  14. Click on Finish recording | Close file | OK, unless a ramp-up frequency or 20 Hz s recordings is being performed.
  15. Finish the recording and save the data by clicking on the Close file and save data button.

3. MVRC analyses

  1. Start the analyzing software program to perform the analysis offline.
  2. Select the recording that will be analysed and click on the OK button.
  3. Click on Load parameters from the Files menu.
  4. Select MANAL9 option for the analysis. If this is not present on the list, click on Browse to find this file. Click OK to continue.
  5. When a description of MAnal9 muscle excitability analysis appears, click OK to continue.
    1. Invert the muscle action potential by typing MM-1 if the potential appears upside down.
    2. Right-click the mouse to make the magenta line visible. Set the window to the base of the peak response and with a width corresponding roughly to the width of the action potential at that height. Drag with the mouse to adjust the window. The window determines the latencies within which the height and latency are measured, as indicated by the pale blue lines, and green line indicates the baseline. Click OK to continue.
  6. Click OK to remeasure the latencies and peaks. This will be done automatically.
    NOTE: In the display of the remeasured latencies, the latencies are measured to shorter delays than original ones. This is because the responses to conditioning stimuli alone were subtracted from responses to the conditioning plus the test. This ensures that conditioning stimuli do not interfere with latency measurements. As is indicated in the prompt box, single bad points can be eliminated by positioning the cursor (vertical red line) over the point and hitting the ~ key. The bad point is replaced with mean of values on either side in same channel. If there are no bad points, set DE (display end) to just after the last latency required.
  7. Click OK to create an RMC file.
  8. Ignore most of the options appearing in the "Create RCC or RMC" form, since these are concerned with measurements of C-fiber rather than MVRCs. Click Save and Exit to continue. After saving the RMC file, the prompt box provides different options
  9. If frequency ramp and/or repetitive stimulation data have been recorded, follow the instructions to analyse these. Otherwise, select Go straight to create MEM file option to create a MEM file. Click OK to continue.
  10. Click Save and Exit to continue.
  11. Click OK to add the RMC data to MEM file.
  12. Click Add from Input RMC file to add this data to the MEM file, then change the directory to save the composite MEM file. Then, click Save and Exit to save it.
  13. Click OK to save the remeasured QZD file to allow differentiation from the original QZD file using a # sign.

Subscription Required. Please recommend JoVE to your librarian.

Representative Results

The following results were obtained in a subgroup of patients from a recent study22, in which there were fibs/psws in all sites showing profuse denervation activity. The results showed that changes in muscle fibers after denervation were assessed in vivo using the MVRC technique described in this protocol. MVRCs showed changes consistent with depolarization of the resting membrane potential in the neurogenic muscle fibers.

Fourteen patients were compared with 29 healthy subjects. Subject demographics are shown in Table 1. Figure 2 illustrates recordings from a healthy subject and patient. Figure 3 and Table 2 illustrate comparison of patients' MVRCs with healthy subjects. MRRP was prolonged, and ESN and LSN were reduced in patients compared to healthy controls (Table 2, Figure 3).

Figure 1
Figure 1: Picture of MVRCs set-up. (A) Isolated linear bipolar constant-current stimulator, (B) 50 Hz noise eliminator, (C) isolated EMG amplifier, and (D) analogue-to-digital converter. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Examples of MVRC recordings. Recordings after one conditioning stimulus (red), two conditioning stimuli (green), and five conditioning stimuli (blue) from a (A) healthy subject and (B) patient with L5 radiculopathy. Please click here to view a larger version of this figure.

Figure 3
Figure 3: MVRCs with one, two, and five conditioning stimuli. (A) MVRCs in 14 patients (grey lines) compared to mean value of 29 healthy controls (filled black squares). Graphical representation of percentage change in latency is plotted against ISIs from 2–1,000 ms (logarithmic scale). (B,C): Same as (A), but with two and five conditioning stimuli. Please click here to view a larger version of this figure.

Healthy controls
Age (years) 55.7 ± 14.9 58.9 ± 16.3
Gender (M/F) 14/15 9/5
Disease duration (months) - 3.4 ± 2.7
MRC score - 3.0 ± 1.1
Etiology - Peronal neuropathy (9)
L5 root afflication

Table 1: Demographics and clinical characteristics. Values are listed as means ± standard deviation. This table has been modified from Witt et al.22.

Healthy Controls
p-value for t-test
MRRP (ms) 3.5 ± 0.4 7.6 ± 3.1 p = 6.8-8
ESN (%) 11.3 ± 2.1 7.6 ± 2.3 p = 5.5-5
ESN (ms) 7.8 ± 1.3 12.7 ± 2.5 p = 1.6-8
5ESN (%) 13.7 ± 2.5 1.0 ± 0.6 p = 9.3-10
LSN (%) 4.1 ± 1.4 2.8 ± 1.7 p = 0.017
XLSN (%) 2.9 ± 0.7 1.0 ± 1.6 p = 1.8-10
5XLSN (%) 8.0 ± 1.4 2.8 ± 1.6 p = 2.2-11

Table 2: Comparison of MVRC parameters between healthy controls and patients. MRRP = muscle relative refractive period; ESN (%) = latency reduction of muscle action potential after one conditioning stimulus as percentage of unconditioned stimulus at ISI of <15 ms. ESN (ms), ISI corresponding to ESN (%). 5ESN = peak early supernormality after five conditioning stimuli. LSN (%) = latency reduction of muscle action potential after one conditioning stimulus as percentage of unconditioned stimulus at ISI between 100–150 ms. XLSN (%) = latency reduction of muscle action potential after two conditioning stimuli as percentage of one conditioning stimulus at ISI between 100–150 ms. 5XLSN (%) = latency reduction of muscle action potential after five conditioning stimuli as percentage of one conditioning stimulus at ISI between 100–150 ms. Values are listed as means ± standard deviation.

Subscription Required. Please recommend JoVE to your librarian.


MVRCs, as programmed in the recording software, is a highly automated procedure, but care is needed to obtain reliable results. In the recording stage, while adjusting the needles, it is important to avoid stimulating the end-plate zone or nerve. This usually leads to large twitches of the whole muscle, which increases the risk of displacement of the stimulation and/or recording needle during recording MVRCs. To date, the method has been applied to several muscles that have better described end-plate zone; however, the endplates may be scattered (i.e., in the anterior tibial muscle). Therefore, particular attention is required.

In order to avoid stimulation of the endplate or nerve instead of muscle fibers, care should be taken when observing the muscle for twitches. The stimulating monopolar needle should be moved, as well as the recording concentric needle, to locate a site that does not cause twitches. Additionally, subjects should be asked whether or not they feel pain. MVRC recordings do not cause any unpleasantness, unless the end-plate zone or the nerve is stimulated instead of muscle fibers.

A limitation of the MVRCs method is performing the recording in only one site and examination of only a few muscle fibers, which does not necessarily represent the whole muscle. This limitation is particularly important in disorders where the pathology is not diffuse. A previous study found surprisingly no difference between patients with amyotrophic lateral sclerosis and healthy controls despite denervated muscles. This was probably because denervation activity was not recorded at the site where MVRCs were recorded23. It also cannot be excluded that the needle could have been adjusted to a healthier spot with a more optimal response.

Another limitation of MVRCs is that one may have a tendency to spot the healthy muscle fibers while adjusting the recording needle to obtain a stable response for measurements. One way to overcome this limitation may be to do the recordings from polyphasic potentials. However, this may pose problems for determining an accurate latency if there are undifferentiated peaks. Additionally, although we intend to stimulate and record from the same bundle of muscle fibers, these may not be exactly the same. The stimulated bundle may contain different fibers during ongoing experiment24.

MVRCs provide information that cannot be obtained by the conventional electrophysiological methods. Thus, there is no other method in current use that can be compared to MVRCs. The earlier report6, using single fiber needle electrodes to record at two sites from the same muscle fiber, was much more difficult. Good recordings were only obtained from 43 out of 118 muscle fiber studies, and this method has not been adopted in research labs or clinics. Another similar but unautomated approach used eight different ISIs from 20 ms to 2 ms25. The authors reported that a recording took 20–60 min, whereas this method records MVRCs with 34 ISIs in about 10 min. The analysis is also fast and highly automated.

In conclusion, MVRCs is a method that may provide invaluable information to understand the underlying mechanisms of neuromuscular disorders. For patients in which a mutation in an ion channel gene has been identified, this method also provides data on the effects of those specific mutations on muscle membrane excitability in vivo. This, together with in vitro expression studies, enables a more accurate understanding of muscle pathophysiology in these patients. This method has the potential to provide insight into the role of those channels in normal muscle physiology, thus improving the understanding of muscle disease in general. Further studies with other patient groups and larger groups are necessary. Studies recording MVRCs in different muscles are also warranted.

Subscription Required. Please recommend JoVE to your librarian.


H.B. receives royalties from UCL for sales of his Qtrac software used in this study. The other authors have no potential conflicts of interest. All authors have approved the final article.


This study was financially supported mainly by the two grants from Lundbeck Foundation (Grant number R191-2015-931 and Grant number R290-2018-751). Additionally, the study was financially supported by Novo Nordisk Foundation Challenge Programme (Grant number NNF14OC0011633) as part of the International Diabetic Neuropathy Consortium.


Name Company Catalog Number Comments
50 Hz Noise Eliminator Digitimer Ltd Humbug
Analogue-to-Digital Converter National Instruments NI-6221
Analysing software program Digitimer Ltd (copyright Institute of Neurology, University College, London) QtracP, MANAL9
Disposable concentric needle electrode, 25 mm x 30G Natus Dantec DCN
Disposable monopolar needle electrode, 25 mm x 26G Natus TECA elite
Isolated EMG amplifier Digitimer Ltd D440
Isolated linear bipolar constant-current stimulator Digitimer Ltd DS5
Software and recording protocol Digitimer Ltd (copyright Institute of Neurology, University College, London) QtracW software, M3REC3 recording protocol written by Hugh Bostock, Istitute of Neurology, London, UK)



  1. Tankisi, H., et al. Pathophysiology inferred from electrodiagnostic nerve tests and classification of polyneuropathies. Suggested guidelines. Clinical Neurophysiology. 116, (7), 1571-1580 (2005).
  2. Gregorio, C. C., Hudecki, M. S., Pollina, C. M., Repasky, E. A. Effects of denervation on spectrin concentration in avian skeletal muscle. Muscle and Nerve. 11, (4), 372-379 (1988).
  3. Kotsias, B. A., Venosa, R. Role of sodium and potassium permeabilities in the depolarization of denervated rat muscle fibers. Journal of Physiology. 392, 301-313 (1987).
  4. Kirsch, G. E., Anderson, M. F. Sodium channel kinetics in normal and denervated rabbit muscle membrane. Muscle and Nerve. 9, (8), 738-747 (1986).
  5. Stalberg, E. Propagation velocity in human muscle fibers in situ. Acta Physiologica Scandinava Supplementum. 287, 1 (1966).
  6. Mihelin, M., Trontelj, J. V., Stalberg, E. Muscle fiber recovery functions studied with double pulse stimulation. Muscle and Nerve. 14, (8), 739-747 (1991).
  7. Z'Graggen, W. J., Bostock, H. Velocity recovery cycles of human muscle action potentials and their sensitivity to ischemia. Muscle and Nerve. 39, (5), 616-626 (2009).
  8. Bostock, H., Tan, S. V., Boerio, D., Z'Graggen, W. J. Validity of multi-fiber muscle velocity recovery cycles recorded at a single site using submaximal stimuli. Clinical Neurophysiology. 123, (11), 2296-2305 (2012).
  9. Z'Graggen, W. J., Troller, R., Ackermann, K. A., Humm, A. M., Bostock, H. Velocity recovery cycles of human muscle action potentials: repeatability and variability. Clinical Neurophysiology. 122, (11), 2294-2299 (2011).
  10. Lee, J. H. F., Boland-Freitas, R., Ng, K. Sarcolemmal excitability changes in normal human aging. Muscle and Nerve. 57, (6), 981-988 (2018).
  11. Lee, J. H. F., Boland-Freitas, R., Ng, K. Physiological differences in sarcolemmal excitability between human muscles. Muscle and Nerve. 60, (4), 433-436 (2019).
  12. Humm, A. M., Bostock, H., Troller, R., Z'Graggen, W. J. Muscle ischaemia in patients with orthostatic hypotension assessed by velocity recovery cycles. Journal of Neurology, Neurosurgery and Psychiatry. 82, (12), 1394-1398 (2011).
  13. Z'Graggen, W. J., et al. Velocity recovery cycles of human muscle action potentials in chronic renal failure. Clinical Neurophysiology. 121, (6), 874-881 (2010).
  14. Z'Graggen, W. J., et al. Muscle membrane dysfunction in critical illness myopathy assessed by velocity recovery cycles. Clinical Neurophysiology. 122, (4), 834-841 (2011).
  15. Lee, J. H., Boland-Freitas, R., Liang, C., Ng, K. Sarcolemmal depolarization in sporadic inclusion body myositis assessed with muscle velocity recovery cycles. Clinical Neurophysiology. 19, (31205-2), 1388 (2019).
  16. Tan, S. V., Z'Graggen, W. J., Hanna, M. G., Bostock, H. In vivo assessment of muscle membrane properties in the sodium channel myotonias. Muscle and Nerve. 57, (4), 586-594 (2018).
  17. Tan, S. V., et al. In vivo assessment of muscle membrane properties in myotonic dystrophy. Muscle and Nerve. 54, (2), 249-257 (2016).
  18. Tan, S. V., et al. Membrane dysfunction in Andersen-Tawil syndrome assessed by velocity recovery cycles. Muscle and Nerve. 46, (2), 193-203 (2012).
  19. Tan, S. V., et al. Chloride channels in myotonia congenita assessed by velocity recovery cycles. Muscle and Nerve. 49, (6), 845-857 (2014).
  20. Boland-Freitas, R., et al. Sarcolemmal excitability in the myotonic dystrophies. Muscle and Nerve. 57, (4), 595-602 (2018).
  21. Stalberg, E., et al. Standards for quantification of EMG and neurography. Clinical Neurophysiology. 130, (9), 1688-1729 (2019).
  22. Witt, A., et al. Muscle velocity recovery cycles in neurogenic muscles. Clinical Neurophysiology. 130, (9), 1520-1527 (2019).
  23. Kristensen, R. S., et al. MScanFit motor unit number estimation (MScan) and muscle velocity recovery cycle recordings in amyotrophic lateral sclerosis patients. Clinical Neurophysiology. 130, (8), 1280-1288 (2019).
  24. Marrero, H. G., Stalberg, E. V. Optimizing testing methods and collection of reference data for differentiating critical illness polyneuropathy from critical illness MYOPATHIES. Muscle and Nerve. 53, (4), 555-563 (2016).
  25. Allen, D. C., Arunachalam, R., Mills, K. R. Critical illness myopathy: further evidence from muscle-fiber excitability studies of an acquired channelopathy. Muscle and Nerve. 37, (1), 14-22 (2008).



    Post a Question / Comment / Request

    You must be signed in to post a comment. Please or create an account.

    Usage Statistics