ORIGINAL RESEARCH published: 19 June 2020 doi: 10.3389/fneur.2020.00541 Depletion of ATP Limits Membrane Excitability of Skeletal Muscle by Increasing Both ClC1-Open Probability and Membrane Conductance Pieter Arnold Leermakers 1*, Kamilla Løhde Tordrup Dybdahl 1 , Kristian Søborg Husted 1 , Anders Riisager 1 , Frank Vincenzo de Paoli 1 , Tomàs Pinós 2,3 , John Vissing 4 , Thomas Oliver Brøgger Krag 4 and Thomas Holm Pedersen 1 1 Department of Biomedicine, Aarhus University, Aarhus, Denmark, 2 Mitochondrial and Neuromuscular Disorders Unit, Vall d’Hebron Institut de Recerca, Universitat Autònoma de Barcelona, Barcelona, Spain, 3 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain, 4 Department of Neurology, Rigshospitalet, Copenhagen Neuromuscular Center, University of Copenhagen, Copenhagen, Denmark Activation of skeletal muscle contractions require that action potentials can be excited and propagated along the muscle fibers. Recent studies have revealed that muscle fiber excitability is regulated during repeated firing of action potentials by cellular signaling Edited by: systems that control the function of ion channel that determine the resting membrane Jean-François Desaphy, University of Bari Aldo Moro, Italy conductance (Gm ). In fast-twitch muscle, prolonged firing of action potentials triggers a Reviewed by: marked increase in Gm , reducing muscle fiber excitability and causing action potential Christoph Fahlke, failure. Both ClC-1 and KATP ion channels contribute to this Gm rise, but the exact Helmholtz-Verband Deutscher Forschungszentren (HZ), Germany molecular regulation underlying their activation remains unclear. Studies in expression Bruno Allard, systems have revealed that ClC-1 is able to bind adenosine nucleotides, and that low Université Claude Bernard adenosine nucleotide levels result in ClC-1 activation. In three series of experiments, Lyon 1, France this study aimed to explore whether ClC-1 is also regulated by adenosine nucleotides in *Correspondence: Pieter Arnold Leermakers native skeletal muscle fibers, and whether the adenosine nucleotide sensitivity of ClC-1 pal@biomed.au.dk could explain the rise in Gm muscle fibers during prolonged action potential firing. First, whole cell patch clamping of mouse muscle fibers demonstrated that ClC-1 activation Specialty section: This article was submitted to shifted in the hyperpolarized direction when clamping pipette solution contained 0 mM Neuromuscular Diseases, ATP compared with 5 mM ATP. Second, three-electrode Gm measurement during muscle a section of the journal fiber stimulation showed that glycolysis inhibition, with 2-deoxy-glucose or iodoacetate, Frontiers in Neurology resulted in an accelerated and rapid >400% Gm rise during short periods of repeated Received: 28 February 2020 Accepted: 14 May 2020 action potential firing in both fast-twitch and slow-twitch rat, and in human muscle Published: 19 June 2020 fibers. Moreover, ClC-1 inhibition with 9-anthracenecarboxylic acid resulted in either an Citation: absence or blunted Gm rise during action potential firing in human muscle fibers. Third, Leermakers PA, Dybdahl KLT, Husted KS, Riisager A, de Paoli FV, Gm measurement during repeated action potential firing in muscle fibers from a murine Pinós T, Vissing J, Krag TOB and McArdle disease model suggest that the rise in Gm was accelerated in a subset of fibers. Pedersen TH (2020) Depletion of ATP Together, these results are compatible with ClC-1 function being regulated by the level Limits Membrane Excitability of Skeletal Muscle by Increasing Both of adenosine nucleotides in native tissue, and that the channel operates as a sensor of ClC1-Open Probability and Membrane skeletal muscle metabolic state, limiting muscle excitability when energy status is low. Conductance. Front. Neurol. 11:541. doi: 10.3389/fneur.2020.00541 Keywords: skeletal muscle, ATP, membrane excitability, fatigue, membrane conductance, ClC-1, McArdle disease Frontiers in Neurology | www.frontiersin.org 1 June 2020 | Volume 11 | Article 541 Leermakers et al. Adenosine Nucleotides Control ClC-1 Function INTRODUCTION to the large C-terminal part of the channel, between two cystathionine-β-synthase (CBS) domains (17–19). These studies Skeletal muscle contractions support human overall health, show that adenosine nucleotide binding shifts the activation underlying both body movement and posture, and ventilation. voltage of ClC-1 in the depolarizing direction, which implies The ability to sustain contractile function over time, which that a loss of cellular adenosine levels could inversely cause enables prolonged physical activity during exercise, is both the activation voltage of ClC-1 to shift in the hyperpolarizing highly variable between different muscles and individuals, and direction, leading to an increased open probability at the resting determines the onset of muscle-fatigue (i.e., a state in which membrane potential with consequent loss of muscle excitability. this contractile function becomes impaired and the muscular As ATP, ADP, and AMP had similar effects on ClC-1, the power output gradually declines). Multiple cellular mechanisms combined adenosine nucleotide levels would have to decrease are likely to contribute to fatigue during exercise, depending on for the channel to shift toward an increased opening probability. the type of exercise and type of muscle fibers being recruited by However, it is currently not clear whether ClC-1 is also regulated the nervous system (1). by adenosine nucleotides in native tissue. The energy consumption of skeletal muscle increases Support for ClC-1 as a metabolic sensor in native tissue came dramatically when contracting during exercise, and a correlation from observations in action potential firing muscle fibers, where between the metabolic state and performance of the muscle it was reported that prolonged action potential firing resulted is well-known (2). Exhaustion of the muscular metabolic in substantial ClC-1 activation in rodent fast-twitch muscle, state may introduce fatigue, and excessive muscle fatigue is leading to reduced muscle fiber excitability in combination with a well-recognized symptom in patients suffering from in- action potential excitation- and propagation failure. As this ClC- born metabolic myopathies involved in glycogen breakdown 1 activation was rapidly reversible, this activation likely reflects such as myophosphorylase deficiency (McArdle disease) (3). physiological relevant ClC-1 regulation during skeletal muscle Mechanistically, several steps in the excitation-contraction activity (20). Although ClC-1 is present in, and regulates the coupling that link neuronal command to muscle force membrane conductance of, both fast- and slow-twitch fiber- production are known to be sensitive to fluctuations in types, this substantial action potential firing-induced ClC-1 metabolites during exercise (2, 4). activation was only observed in fast-twitch muscle fibers. As Action potential excitation and propagation are essential steps fast-twitch muscle fibers have been shown to undergo larger in the normal activation of skeletal muscle fibers to generate ATP-depletion during exercise than slow-twitch fibers (21), these muscle contractions, and surface membrane ion channels results are compatible with a possible role of ClC-1 as a metabolic and ion transport proteins are key factors determining the sensor linking metabolic state to muscle fiber excitability. electrophysiological properties and excitability of muscle fibers. The cellular signaling that underlies ClC-1 activation The ClC-1 chloride (Cl− ) ion channel is a voltage-gated chloride during prolonged action potential firing remains incompletely channel (5), that is exclusively expressed in skeletal muscle to understood, and there is no data available describing the physiological relevant levels (5–7). This ion channel, which only relationship between adenosine nucleotides and ClC-1 activation has a single channel conductance of about 1 pS (8), accounts for in native skeletal muscle tissue. The present study aimed 80% of the total ion permeability of the surface membrane of to explore if depletion of adenosine nucleotides results in resting muscle fibers, a feature that is highly conserved among increased ClC-1 opening in native tissue of muscle fibers. Such muscle fiber types and species (9–13). a mechanism of ClC-1 regulation may underlie the ClC-1 The equilibrium potential for Cl− is close to the resting activation during prolonged action potential firing that has been membrane potential (Vm ) in muscle fibers, because Cl− is observed in fast-twitch muscle fibers of rat and mice (20, 22, 23). predominantly passively distributed across the muscle fiber Given that this ClC-1 activation has been observed in muscles surface membrane. Together, these properties of Cl− (i.e., high with a fast-twitch fiber-type only, the present study also explored Cl− membrane conductance (GCl ), passive Cl− distribution, and whether similar rises in membrane conductance can be triggered the equilibrium potential that is close to resting Vm ) enforce in both rat muscle with a slow-twitch fiber-type and in human ClC-1-mediated Cl− currents to stabilize the resting membrane abdominal muscle with a mixed fiber-type under conditions of potential. Therefore, GCl and muscle excitability are inversely compromised glycolysis. Moreover, as a proof-of-principle, this related, with reduction in GCl enhancing muscle fiber excitability study explored whether skeletal muscle fibers of McArdle mice while a rise in GCl dampening excitability. A significant rise in GCl were prone to an accelerated increase in membrane conductance may even compromise muscle excitability and prevent muscle during repetitive action potential firing. fiber activation, indicating that ClC-1 has the capability to serve as a physiological gatekeeper of the first step in the excitation- contraction coupling that underlies muscle activation (14–16). MATERIALS AND METHODS During the last decade, the understanding of the biophysical Animals and Ethical Approval properties of ClC-1, and the cellular signaling systems that C57BL/6 mice (3–4 week old, male/female), Wistar rats (12– regulate ClC-1 expression and function, has been greatly 14 week old, females), and McArdle mice (12 months old, expanded. Interestingly, studies from expression systems suggest male/female) were housed under 12 h light/dark conditions at that adenosine nucleotides (i.e., ATP, ADP, and AMP) regulate 21◦ C and fed ad libitum. Wistar rats and C57BL/6 mice were the voltage dependency of the ClC-1 channel by binding euthanized by CO2 exposure/inhalation, and McArdle mice Frontiers in Neurology | www.frontiersin.org 2 June 2020 | Volume 11 | Article 541 Leermakers et al. Adenosine Nucleotides Control ClC-1 Function were anesthetized with s.c. injection of Hypnorm/Midazolam using a micropipette puller model P-97 (Sutter Instruments, (0.01 ml/g, 25% Hypnorm, 25% Midazolam, 50% H2 O) California, US), and were back-filled with either an intracellular and subsequently euthanized by cervical dislocation. Extensor (IC) solution containing 0 mM ATP [10 mM BAPTA, 180 mM digitorum longus (EDL), flexor digitorum brevis (FDB), or HEPES, 1.3 mM MgSO4 , 5 mM Na2 SO4 , 15 mM CsCl, pH 7.2 soleus (SOL) muscles were freshly harvested for experimental by CsOH (around 95 mM)] or 5 mM ATP [10 mM BAPTA, procedures. Both EDL and FDB contain predominantly fast- 174.5 mM HEPES, 5 mM Na2 ATP, 5.85 mM MgSO4 , 15 mM twitch fibers, while SOL contains predominantly slow-twitch CsCl, pH 7.2 by CsOH (around 90 mM)]. fibers (24–26). To achieve similar free Mg2+ content in both solutions, the McArdle (PYGMR50X/R50X ) and representative wild-type required amount of MgSO4 was calculated with Maxchelator (PYGMwt/wt ) mice were generated on a C57BL/6 background (Chris Patton, Stanford University, US). Muscle contractions and extensively characterized as previously described (27, 28). were prevented by BAPTA (a highly selective Ca2+ chelator) All handling and use of animals complied with Danish Animal ensuring a proper seal during recordings. Moreover, the Welfare regulations and was conducted in accordance with the composition of the IC and EC solutions prevented any K+ European Convention for the Protection of Vertebrate Animals (0 mM K+ , TEA-Cl and Cs+ ) and Ca2+ (nifedepine) currents used for Experimental and Other Scientific Purposes (ETS 123). and allowed only negligible Na+ currents (10 mM Na+ in the Experimental procedures were approved by the animal welfare IC solution). officer at Aarhus University or Copenhagen University, and the complied with Danish Animal Experiments Inspectorate (permit Electrophysiology no 2014-15-0201-00041). ClC-1 current amplitude was determined in individual muscle fibers using the whole-cell voltage clamp technique controlled Human Muscle Fibers and Ethical Approval by a MultiClamp 700B Amplifier (Molecular Devices, California, Human rectus abdominis muscle (HAM) biopsies were isolated US) and sampled using an analog-to-digital Micro1401-3 as described elsewhere (29). In short, bundles of well-defined converter (Cambridge Electronic Design, UK). The voltage muscle fibers with tendinous insertions at both ends, measuring protocol and data acquisition were controlled using Signal 6.03 approx. 8 cm × 2 cm × 1, cm were isolated from human software (Cambridge Electronic Design, UK) with a sampling subjects undergoing for abdominal surgery. Subjects had no frequency of 40 kHz. known neuromuscular diseases. HAM contains an almost equal The glass pipettes were inserted in the experimental bath contribution of fast- and slow-twitch fibers (30). The isolation under a slight positive pressure. At first contact between the and use of human skeletal muscle was approved by the Danish pipette and the fiber membrane, the positive pressure was Ethics Committee, Region Midtjylland, Comité I (reference released, and a negative pressure was generated to establish a tight number 1-10-72-20-13), and experiments were performed in seal between the glass pipette and the membrane. A minimum accordance with the Declaration of Helsinki. Informed consent resistance of 1.5 G was obtained before going whole-cell. To was obtained from test subjects. secure proper equilibration of the IC solution, no recordings were made for the first 8 min after establishing whole cell Whole Cell Patch Clamping voltage control. Experiments were only possible in fibers where Muscle Preparation contractions were abolished by the BAPTA that was diffused C57BL/6 FDB muscles were incubated in HEPES-Ringer solution into the muscle fibers. Hence only fibers where dialysis of the [122 mM NaCl, 15 mM Na-HEPES, 9 mM HCl, 2.8 mM KCl, intracellular space had taken place could be used. During the 1.27 mM CaCl2 , 1.2 mM MgSO4 , 1.2 mM KH2 PO4 , 5 mM D- recordings, the serial resistance was compensated to at least glucose and collagenase type 1 (2 mg/ml)] for 45 min at 37◦ C. 90%. The voltage protocol started at −40 mV (holding potential), Collagenase was removed by a serial removal of supernatant and consisted of multiple cycles that each contained a 200-ms followed by washing in solution without collagenase. To depolarization step (+60 mV), a 250-ms variable “test” voltage dissociate the individual fibers, the muscles were triturated with step (between +60 to −140 mV), and a 200-ms constant step Pasteur pipettes of decreasing pore size, and only fibers with (−100 mV) (Figure 1A) from which the instantaneous current maximal length of 400 µm and clear striation were used for or tail current was obtained. This is a similar procedure to experiments. Fibers were stored in HEPES-Ringer solution at what has been used elsewhere (31). The holding potential of 5◦ C, and all electrophysiological recordings were performed −40 mV corresponds to the calculated equilibrium potential for within 10 h after dissection. Cl− under the experimental conditions and this meant that ClC- 1 current would be minimal at the holding potential. Some Experimental Setup leak current did develop over the course of the experiments The dissociated FDB fibers were mounted in an organ bath with that at least in part could reflect incomplete ClC-1 deactivation a constant perfusion of extracellular (EC) solution [72.5 mM between sweeps. This was corrected for in the data analysis. TEA-Cl, 1.2 mM CaCl2 , 6 mM MgSO4 , 120 mM HEPES, 20 µM The extracellular Cl− concentration, which is lower relative to nifedepine, pH 7.3 by CsOH (around 40 mM)], and they more physiological levels (74.9 vs. ∼140 mM), was chosen to were allowed to adhere to the glass surface for 5 at least minimize ClC-1 current to improve voltage control during the minutes before perfusion of chamber was started. Glass patch voltage steps. The variable “test” voltage started at −140 mV in pipettes, with a maximum resistance of 2.5 M, were pulled the first cycle and increased with 10 mV with each cycle. Three Frontiers in Neurology | www.frontiersin.org 3 June 2020 | Volume 11 | Article 541 Leermakers et al. Adenosine Nucleotides Control ClC-1 Function A 0 mM ATP D E 60 -100 ** V(mV) -40 n=5 1.0 -100 -140 -80 0.8 ClC-1 open probability B 40 -60 V½ (mV) 20 0.6 0 0.4 n=3 -40 I(nA) -20 0.2 -40 -20 5mM ATP -60 0.0 0 mM ATP 0 C -150 -100 -50 0 50 5 0 mM ATP 10 Membrane potential (mV) I(nA) 0 -10 0.25 0.50 0.75 sec FIGURE 1 | ATP depletion results in an increased ClC-1 open probability. Isolated murine fast-twitch FDB muscle fibers were subjected to whole-cell patch clamping. (A) The protocol used to evoke currents through ClC-1 in fibers exposed to pipette solution with 0 or 5 mM ATP. (B,C) Examples of whole-cell current traces from a fiber before (B) and after (C) incubation with 400 µM 9-AC. (D) The average open probability of ClC-1 with either 0 or 5 mM ATP in the pipette. Data are depicted as average ± SD and the solid line represents a sigmoidal fit to the data. (E) The average V½ for fibers exposed to pipette solution with 0 or 5 mM ATP. A T-test was performed to test significance of observed differences between 0 mM ATP (n = 5) and 5 mM ATP (n = 5) and p < 0.01** is depicted. repeats were performed at each test voltage. In a few fibers, the Two- and Three-Electrode Membrane test voltage started at −100 mV but this did not appear to affect Conductance Measurements in Action the average data and it was judged acceptable to include all data. Potential Firing Muscle Fibers Measurements were performed first in absence and subsequent Experimental Setup in presence of 400 µM 9-anthracenecarboxylic acid (9-AC). Leak Freshly excised skeletal muscles from Wistar rat (EDL or current was subtracted in all recordings, and having recordings SOL), mice (McArdle mouse EDL), or human (HAM) were from the same fibers before and after 9-AC meant that residual used in these experiments. In all cases, the muscles were leak-current and capacitive artifact could be eliminated by mounted under slight tension in an organ bath containing Krebs- subtracting the measurement with 9-AC from the measurement Ringer solution (122 mM NaCl, 25 mM NaHCO3 , 2.8 mM KCl, without 9-AC. The 9-AC sensitive current was taken to represent 1.2 mM KH2 PO4 , 1.2 mM MgSO4 , 1.3 mM CaCl2 ). The solution ClC-1 current. was supplemented with 5.0 mM D-glucose (Sigma Aldrich, The open probability (Po ) of ClC-1 at the different clamping Denmark) (CTRL), 5.0 mM 2-deoxy-D-glucose (2DG, Sigma voltages was estimated from the peak tail currents at the Aldrich, Denmark)/100 IU insulin (Humulin R, Lilly, USA), or −100 mV constant voltage step that followed the variable 5.0 mM D-glucose + 100 µM iodoacetate (IAA, Sigma Aldrich, test voltage steps (Figures 1A–C). By comparing the peak tail Denmark) as indicated. For experiments with 2DG, muscles currents that followed each test voltage step to the tail current were exposed to 2DG/insulin for at least 3 h before experiments obtained when the test pulse was +60 mV, the open probability were initiated. This was done to enable the proper loading of (Po ) could be calculated and plotted against the clamping voltage 2DG as facilitated by insulin into the muscle fibers to impose step. Based on these calculations, the data from each fiber was the metabolic challenge of compromise glycolytic flux. Other fitted to the 4-parameter sigmoidal function (Equation 1): muscles were incubated in the organ bath for at least 20 min prior to initiating experiments. Pharmacological inhibition of a ClC-1 was achieved by supplementing HAM muscle with 100 µM Po(V) = Po(r) + V −V (1) 9-Anthracenecarboxylic acid (9-AC, Sigma Aldrich, Denmark). 1 + exp( ½b ) 10 nM tetrodotoxin (TTX, Tocris, UK) was used to prevent spontaneous action potential firing in the presence of 9-AC. The Where Po(V) is the channel open probability at the voltage V, buffer was continuously gassed with a mixture of 95% O2 /5% Po(r) is a constant that represents the residual channel opening at CO2 (pH ≈ 7.4) and kept at 30◦ C. Glass pipettes (8–12 M) were infinitely hyperpolarized conditions, a represents the amplitude pulled using a micropipette puller model P-97 and back-filled from the residual current to the maximal ClC-1 current at with 2 M potassium-citrate. +60 mV (maximal Po ), b represents the slope, and V½ represents To enable electrodes to remain inserted in the muscle fibers the midpoint potential at which 50 % of a is achieved. while repeatedly firing action potentials, the contractile activity Frontiers in Neurology | www.frontiersin.org 4 June 2020 | Volume 11 | Article 541 Leermakers et al. Adenosine Nucleotides Control ClC-1 Function of the muscles was reduced by 50 µM N-benzyl-p-toluene Gm , the surface membrane specific conductance, was then sulphonamide (BTS, Toronto Research Chemicals, Canada) for calculated by dividing gm with the surface area per unit length EDL (32), and 25 µM blebbistatin (Sigma Aldrich, Denmark) for of fiber (FSA). In resting muscle fibers a common approach SOL or HAM. Previous studies have shown that these compounds for estimating FSA is to first determine the cross sectional area have minimal effect on muscle excitability (20) and BTS was of the fiber (CSA) from ri assuming a constant value for the shown to reduce energy consummation during activity only by specific sarcoplasmic resistivity (Ri ), here taken to be 180  cm 20% (33). BTS and blebbistatin were dissolved in DMSO, which as determined in resting fibers (35), using Equation 5: resulted in a maximal DMSO concentration of 0.15%, which did not affect resting conditions in muscles. BTS and blebbistatin Ri were added to the solution that perfused the experimental ri = (5) CSA chamber at least 30 min prior to start of measurements, and reduction in contractile activity was visually verified. Next, by assuming the muscle fiber to be a perfect cylinder, FSA Individual muscle fibers and the electrodes were visualized can be calculated from CSA as: using a Nikon DS-Vi1/Nikon Eclipse FN1 microscope. Electrodes were connected to TEC-05X clamp amplifier systems (NPI, √ Germany), and to a Power1401 converter (Cambridge Electronic FSA = 4πCSA (6) Design, UK). The voltage protocol and data acquisition were controlled using Signal software (Cambridge Electronic Design, and Gm can be then be calculated as: UK) with a sampling frequency of >30 kHz. gm gm Gm = = √ (7) FSA 4πCSA Electrophysiology Electrophysiology was performed using the two-electrode Stimulation protocol mimicking muscle activity using technique for McArdle mice and three-electrode technique for three-electrode technique Wistar rats and human muscle biopsy material as described Repeated firing of action potentials with interspaced Gm previously (23). determinations was performed in muscle fibers from Wistar rat (SOL and EDL) and human (HAM) muscles with the three- Gm determination using three electrodes electrode technique. Three electrodes were inserted in these Three electrodes (E1 -E3 ) were placed into the same fiber, where muscle fibers, as described above under Gm determination, and the inter-electrode distance between E2 -E3 (X1 ) was twice the current was injected according to a current-injection protocol inter-electrode distance between E1 -E2 (X2 , Figure 2A). Current with a pre-defined number of cycles. Each cycle of the protocol (I) was first, injected by E1 while the steady membrane potential started with two square negative currents (−30 nA, 50 ms) which response (1V) was measured with E2 and E3 , and subsequently, were injected through E1 and E3 , respectively (used for the Gm current was injected by E3 while 1V was measured with E1 and determination as described above), followed by either a train of E2 . This resulted in the measurement of 1V at three different action-potential generating positive currents injected through E1 distances on the fiber (X1 -X3 ). The transfer resistances at the (i.e., stimulation protocol) or a rest period for the remainder of different inter-electrode distances were calculated by dividing the cycle (i.e., recovery protocol). All three electrodes recorded 1V by I, and they were plotted against the respective inter- the membrane potential at their respective locations. electrode distances and, finally fitted to the cable equation For Wistar rat SOL/EDL fibers, the stimulation protocol (Equation 2) that applies to cells of infinite length with a [1000 ms, 9 pulses (500 nA, 15 Hz, 1 ms)] was repeated 500x to membrane represented by a parallel RC circuit (34). Rin and λ trigger 4500 action potentials in control fibers, or 200x to trigger refer to the fiber input resistance and length constant of the fiber 1800 action potentials in 2DG-exposed fibers. Subsequently, that were obtained from the cable equation fit (Equation 2): the recovery protocol [1000 ms, 0 pulses] was repeated 120x during which no action potentials were triggered and only Gm 1V(x) = 1Vx=0 exp(−xλ−1 ) = IRin exp(−xλ−1 ) (2) was monitored, which was followed by a re-stimulation period in which the stimulation protocol was again repeated 120x to 1V(x) represents the steady state change in membrane potential trigger 1080 action potentials. The stimulation duration used in at position x relative to where the current was injected at x = rat muscle fibers was based on a previous publication, which 0. From Rin and λ the membrane resistance per unit length of describes the presence and absence of a rise in Gm in EDL and fiber (rm ), the intracellular resistance per length of muscle fiber SOL in this time-frame, respectively (20). (ri ), and the membrane conductance per unit length of fiber (gm ) For HAM fibers, the stimulation protocol [1400 ms, 10 pulses were calculated: (300 nA, 30 Hz, 1 ms)] was repeated 143x to trigger 1430 action potentials in all conditions. The protocol was ceased before it rm = 2Rin λ = (gm )−1 (3) reached 1430 action potentials when failure in action potential excitation/propagation was observed. The stimulation duration used in HAM fibers was based on the development of the rise in 2Rin Gm under glycolysis inhibition and the protocol is similar to a ri = (4) previous publication (15). λ Frontiers in Neurology | www.frontiersin.org 5 June 2020 | Volume 11 | Article 541 Leermakers et al. Adenosine Nucleotides Control ClC-1 Function A B stimulation / re-stimulation period C recovery period 500 500 E1 E2 E3 E1 I (nA) E1 I (nA) 0 0 muscle fiber -40 -40 X1 X2 0.1 s 0.1 s E3 I (nA) E3 I (nA) X3 40 40 0 0 -40 -40 Square Action potential Square No action potential current triggering currents current triggering currents D E2 E3 ↓ 30 Membrane potential (mV) 0 1st AP train -30 -60 0.1 s 2 ms -90 E E2 E3 ↓ 30 Membrane potential (mV) 0 100th AP train -30 -60 -90 F E2 E3 0 Membrane potential (mV) ↓ -30 500th AP train -60 -90 G E2 E3 potential (mV) Membrane -60 120th recovery period -90 FIGURE 2 | Voltage protocol and representative traces of a rat EDL-muscle control fiber using the three-electrode technique. (A) Schematic depiction of the placement of three electrodes (E1 , E2 , E3 ) in a muscle fiber, in order to obtain three inter-electrode distances (X1 , X2 , X3 ). (B) Schematic depiction of the current injections through E1 and E3 during the Gm determination and action potential stimulation protocol. The used protocol, with a total duration of 1000 ms, consisted of two square hyperpolarizing currents used for determination of Gm (−30 nA, 50 ms duration) and a train of large amplitude depolarizing current pulses to trigger a short train of action potentials [9 pulses (500 nA, 15 Hz, 1 ms)]. (C) The recovery protocol was similar to (B) except that the action potential firing pulses were not included. (D–G) Representative traces of the membrane potential recorded with E2 and E3 in a rat EDL muscle fiber during a CTRL experiment, with the first action potential measured with E3 (black downward arrow) depicted at higher time resolution on the right side. (D) Traces obtained during injection of the first action potential (AP) train of the protocol, (E) during the 100th action potential train of the protocol, and (F) during the 500th action potential train of the protocol. (G) The last (120th) trace of the recovery protocol. Frontiers in Neurology | www.frontiersin.org 6 June 2020 | Volume 11 | Article 541 Leermakers et al. Adenosine Nucleotides Control ClC-1 Function Stimulation protocol mimicking muscle activity using human biopsy data originated from one HAM muscle. Data two-electrode technique was presented as mean ± SEM unless otherwise indicated. The two-electrode technique was chosen for experiments with Differences between data depicted in two groups were tested EDL muscles from McArdle mice as this approach has a higher with Student’s t-test, and differences between data depicted in success rate, and these animals were in very short supply. multiple groups were tested with ANOVA and Bonferroni’s post- Likewise, the stimulation protocol was chosen to maximize the hoc test (parametric distributions) or Kruskal-Wallis and Dunn’s success rate of the experiments. Two electrodes (E1 -E2 ) were post-hoc test (non-parametric distributions) where appropriate. placed in close proximity (≈0 µm) within one fiber, of which E1 Differences between binary data depicted in multiple groups were injected current according to a current-injection protocol with tested using the Fisher’s exact test, and corrected for multiple a pre-defined number of cycles, and E2 measured the membrane comparisons. p<0.05 was considered significant, and p<0.05∗ , potential at x = 0. Each cycle of the protocol started with a square p<0.01∗∗ , p<0.001∗∗∗ are depicted in the figures. negative current (−30 nA, 50 ms), which was used to measure the fiber Rin . This was followed either by a train of action-potential RESULTS generating positive currents (i.e. stimulation protocol) or a rest period for the remainder of the cycle (i.e., recovery protocol). ATP-Dependent ClC-1 Open Probability in The stimulation protocol [1500 ms, 15 pulses (300 nA, 30 Hz, Native Skeletal Muscle Fibers 1 ms)] was repeated 200x to trigger 3000 action potentials. To study whether ClC-1 opening is sensitive to differences Subsequently, the recovery protocol [1500 ms, 0 pulses] was in intracellular ATP levels ([ATP]) in native skeletal muscle repeated 100x during which no action potentials were triggered fibers, whole cell patch clamping was performed on isolated fast- and only Rin was monitored, which was followed by a re- twitch muscle fibers from mice with either 0 or 5 mM ATP stimulation period in which the stimulation protocol was again in the pipette solution. The protocol for determining ClC-1 repeated 100x to trigger 1500 action potentials. The stimulation current in the muscle fibers and the calculation of the voltage was continued until the Rin started to decline drastically reflecting dependent open probability (Po ) of ClC-1 was as performed as the development of the rise in Gm . previously described elsewhere (31) (Figure 1A). Ionic currents These experiments thus measured Rin and to convert this were measured before (Figure 1B) and after addition of 400 µM into Gm during activity, the three electrode techniques described 9-AC (Figure 1C) in the same muscle fiber, and the ClC-1- above in 2.4.2.2. was used to determine Gm in inactive fibers of the specific ionic current was calculated by subtracting the ionic same muscle. Gm during muscle activity could then be estimated current obtained with 9-AC from the current obtained without using equation 8 as described previously (22). 9-AC. In general, the membrane current in the presence of 9- AC was markedly reduced, showing that ClC-1 facilitated the majority of the current flow under these experiment conditions Rin(t = 0) 2 Gm(t=x) = Gm(t = 0) (8) (compare black and gray traces in Figures 1B,C). There was no Rin(t = x) 2 significant difference in peak ClC-1 current at +60 mV between groups exposed to 5 and 0 mM ATP pipette solutions (5 mM Fitting properties to determine the timing and size of the rise ATP: −40 ±13 nA, 0 mM ATP: −34 ±16 nA, p = 0.553). Also, in Gm there was no difference in currents between the groups after Based on observations of Gm during muscle activity the data 9-AC exposure. from each fiber was fitted to the following 4-parameter sigmoidal The calculated Po at each specific membrane potential was function as previously described, to determine the timing and plotted against that membrane potential, and subsequently fitted maximal size of the rise in Gm (22) (Equation 9): to the sigmoidal function in Equation 1 (Figure 1D). The parameters from the fits of individual fibers were obtained and 1Gm(max) Gm = Gm(min) + (9) comparison of parameters was performed between groups. The 1 + exp( txb−t ) slope (5 mM ATP: 33 ±23 mV, 0 mM ATP: 33 ±8 mV, p = 0.944) and residual opening probability at the most hyperpolarized Where Gm represents the Gm at the time t, Gm(min) represents potentials (5 mM ATP: −0.074 ± 0.232, 0 mM ATP: −0.181 ± the minimal Gm , 1Gm(max) represents the difference between 0.319, p = 0.563) were not different between the groups. The the minimal and maximal Gm , b represents the slope, and tx membrane voltage that resulted in 50% of the voltage dependent represents the time at which Gm is 50% of 1Gm(max) . ClC-1 current to be activated, V½ , was shifted significantly in the hyperpolarizing direction in fibers subjected to a pipette solution Statistics and Data Handling with 0 mM ATP (Figures 1D,E). All fibers in which the resting membrane potential recorded by one of the electrodes depolarized beyond −60 mV during A Repetitive Action Potential the experimental procedure were excluded from analyses. The number of analyzed fibers (n) is depicted for each Firing-Induced Rise in Gm Occurs Only in group in the graph legends, and originated from at least 3 Fast- and Not in Slow-Twitch Fibers Under different animals per group for all experiments performed on Conditions With Functional Glycolysis C57BL/6 mice and Wistar rats. Data from McArdle mice and The initial series of experiments in muscle fibers was conducted representative controls were shown per individual mouse, and to verify previous findings that repetitive action potential firing Frontiers in Neurology | www.frontiersin.org 7 June 2020 | Volume 11 | Article 541 Leermakers et al. Adenosine Nucleotides Control ClC-1 Function resulted in large increases in Gm with subsequent loss of at a much lower maximal Gm compared with the maximal Gm muscle excitability in fast-twitch (EDL) but not in slow-twitch of glycolysis-inhibited EDL muscle fibers (Figures 3B–D). Like (SOL) muscle fibers from rat. Experimentally, the three-electrode in glycolysis-inhibited EDL muscle fibers, the Gm of glycolysis- technique (Figure 2A) was used and a 1000 ms stimulation inhibited SOL muscle fibers returned to baseline values in the protocol was repeated 500 times in each fiber. During each recovery period, and subsequently increased quickly upon re- run of the protocol, Gm was first determined with two square stimulation (Figure 3C). currents, and, subsequently, 9 action potentials were triggered in the fiber (Figure 2B). After 500 runs of the protocol, the Inhibition of Glycolysis Triggers an Action action potential firing was ceased, and the recovery of Gm was Potential Firing-Induced ClC-1-Dependent monitored during 120 runs of the protocol without triggering action potentials (Figure 2C). Finally, action potential triggering Rise in Gm in Human Muscle Fibers Previous experiments, using a similar approach to determine Gm was re-started and 120 runs of the action potential triggering changes during repetitive action potential firing, have shown that protocol (Figure 2B) were performed. although ClC-1 inhibition during the first phase of activity is In line with previous work from our group, Gm showed a also present in human muscle, a prolonged activity-induced rise biphasic response during repetitive action potential stimulation in Gm has not been observed in the first 2000 action potentials in EDL but not in SOL muscle fibers (15, 20, 22). Thus, (15). To determine whether this rise in Gm can, similar to SOL Gm decreased during the first 1500 action potentials (phase muscles from rat, be brought out with inhibition of glycolysis, 1) of repetitive action potential firing in both EDL and SOL a series of experiments with the three-electrode technique was muscle fibers (Figures 2E, 3A). This has previously been shown conducted in HAM muscle in the absence and presence of to primarily reflect protein kinase C-mediated inhibition of iodoacetate (IAA). Indeed, no repetitive action potential firing- ClC-1 ion channels in both muscle fiber types (15, 20), and induced rise in Gm was observed in control conditions in was not the aim of the current manuscript. In EDL fibers human muscle fibers during the 200 s of running the stimulation only, Gm rapidly increased after around 1500–2000 action protocol (Figures 4A,D,G). Similar to the results from glycolysis- potentials (phase 2) (Figures 2F, 3A). Gm returned to baseline inhibited rat SOL muscle fibers, however, inhibition of glycolysis values during the recovery period, and subsequently increased resulted in the appearance of a phase 2-like increase in Gm in quickly upon re-stimulation (Figures 2G, 3A). During prolonged all tested human muscle fibers (Figures 4B,E,G). This increase action potential firing, the action potential amplitude, and in Gm correlated strongly with action potential-excitation failure, depolarization and repolarization speed decreased gradually over as can been clearly seen in the action potential train from the time (Figures 2D–F). representative trace depicted in the third panel of Figure 4B. To study the relative contribution of ClC-1 to the observed increase The Repetitive Action Potential in Gm , the above described glycolysis-inhibition experiments Firing-Induced Rise in Gm Occurs in Both were repeated in presence of the ClC-1 inhibitor 9-AC. ClC- Fast- and Slow-Twitch Fibers Under 1 inhibition prevented the rise in Gm in 75% of the measured glycolysis-inhibited human muscle fibers (Figures 4C,F,G). As Conditions of Compromised Glycolysis ClC-1 inhibition resulted in a binary effect on the rise of To test if the regulation of Gm is dependent on a functional Gm , measured fibers were depicted individually and not as ATP supply, pharmacological inhibition of glycolysis (i.e., by mean values (Figures 4D–F), and the percentage of fibers replacing 5.0 mM D-glucose with 5.0 mM 2DG + 100 IU that showed an increase in Gm during the first 200 s of insulin) was performed in the above-described experiments, the stimulation protocol were depicted (Figure 4G). During but with a shorter action potential stimulation period (200 s, prolonged action potential firing, the action potential amplitude, 1800 action potentials). The stimulation period was shorter in and depolarization and repolarization speed decreased gradually these experiments because less action potentials were needed to over time. Moreover, 9-AC-mediated inhibition of ClC-1 trigger the activity-induced rise in Gm . Glycolysis-inhibited EDL currents resulted in a decreased repolarization speed at baseline muscle fibers showed an accelerated phase 2-like increase in Gm (Figures 4A–C). Since the access to human muscle was limited, directly after the onset of action potential-firing (Figures 3B,E). IAA was used to inhibit glycolysis, as this approach was much Moreover, this rise in Gm was steeper and reached a higher faster and allowed paired measurements to be performed in the maximal Gm before becoming stable compared to muscle with same preparations. This was not possible with 2DG that required fully functional glycolysis (Figures 3B,D). Consequently, an substantial pre-incubation. initial phase 1-like decrease in Gm was not observed in glycolysis- inhibited EDL muscle fibers. Similar to control conditions, Gm returned to baseline values in the recovery period, and Action Potential Firing-Induced Rise in Gm subsequently increased quickly upon re-stimulation (Figure 3B). Is Accelerated in Murine McArdle EDL Similar to the results from glycolysis-inhibited EDL muscle Muscle Fibers fibers, inhibition of glycolysis resulted in a rapid phase 2-like The above-described experiments in rat and human muscles increase in Gm in SOL muscle fibers (Figures 3C,E). Moreover, showed that Gm rise can be markedly accelerated by the increase in Gm in glycolysis-inhibited SOL muscle fibers was pharmacological inhibition of glycolysis. This is compatible also steep but plateaued earlier (±450 action potentials), and with ClC-1 being a metabolic sensor that can switch open Frontiers in Neurology | www.frontiersin.org 8 June 2020 | Volume 11 | Article 541 Leermakers et al. Adenosine Nucleotides Control ClC-1 Function A B 3500 6000 EDL(CTRL) EDL(CTRL) 3000 SOL(CTRL) EDL(2DG) 5000 Membrane conductance (mS/cm2) Membrane conductance (mS/cm2) 2500 4000 2000 3000 1500 2000 1000 500 1000 0 0 0 100 200 300 400 500 600 700 0 100 200 300 400 500 600 700 Time (s) Time (s) 0 900 1800 2700 3600 4500 Action potentials 0 900 1800 2700 3600 4500 Action potentials C 3500 SOL(CTRL) 3000 SOL(2DG) D E CTRL Membrane conductance (mS/cm2) 2DG 2500 6000 *** 450 4000 2000 *** *** 5000 375 *** tx(action potentials) conductance (mS/cm2) Maximal membrane 3000 1500 4000 300 tx (s) 3000 225 2000 1000 *** 2000 150 500 1000 1000 75 * 0 0 0 nd 0 0 100 200 300 400 500 600 700 EDL SOL EDL SOL Time (s) 0 900 1800 2700 3600 4500 Action potentials FIGURE 3 | Changes in Gm during repetitive action potential firing in fast- and slow-twitch fibers under control conditions and with inhibition of glycolysis. (A) Gm of EDL (black line) and SOL (gray line) muscle fibers in buffer with 5.0 mM D-glucose is depicted over time. Gm of EDL (B) and SOL (C) muscle fibers in solution with 5.0 mM D-glucose (dark line) or 5.0 mM 2DG + 100 IU insulin (light-gray line) is depicted over time. Gm(max) (D) and time until 50% of Gm(max) is reached (tx ) (E) were extracted from sigmoid-fitted data and depicted. CTRL fibers were subjected to 500x the stimulation protocol [1000 ms, 9 pulses (500 nA, 15 Hz, 1 ms)], 120x the recovery protocol [1000 ms, 0 pulses], and to a re-stimulation period that consisted of 120x the stimulation protocol. 2DG fibers were subjected to 200x the stimulation protocol, 120x the recovery protocol, and to a re-stimulation period that consisted of 120x the stimulation protocol. Data from EDL-CTRL and SOL-CTRL fibers depicted in panel (A) is also depicted as CTRL data in panel (B,C), respectively. Gray vertical bars represent the recovery period during the stimulation protocol. ANOVA with Bonferroni’s post-hoc tests were performed to test significance of observed differences between EDL-CTRL (n = 4), EDL-2DG (n = 12), SOL-CTRL (n = 18), and SOL-2DG (n = 32), and p < 0.05* or p < 0.001*** are depicted. and compromise muscle excitability if the metabolic state of fiber. The muscle fibers from the first McArdle mice showed a muscle fibers becomes critical. To further explore for a link consistently accelerated increase in Gm compared with wild-type between membrane conductance and glycolysis, a genetic muscle fibers. However, the muscle fibers from the second model for impaired muscle glucose and energy homeostasis McArdle mouse did not show an accelerated increase in Gm (i.e., McArdle mouse model) was used. Interestingly, the compared with wild-type (Figures 5A,B). Both McArdle and observations from the two analyzed McArdle mice showed wild-type muscle fibers showed a similar return of Gm to baseline marked differences regarding the onset of the repetitive action during the recovery period as well as a similar rapid increase potential firing-induced rise in Gm (Figure 5A). Experimentally, in Gm upon re-stimulation (Figure 5A). Taken together, the the two-electrode technique was used and a 1500 ms stimulation combined observations are compatible with malfunctional protocol was repeated 200 times (300 sec, 3000 action potentials), glycolysis (pharmacologically or genetically induced) being followed by a recovery period (150 s, 0 action potentials), and a able to introduce an accelerated rise in Gm during muscle re-stimulation period in which the same stimulation protocol activity. Given the limited amount of data available from these was repeated 100 times (150 s, 1,500 action potentials) in each McArdle mice, this data must be considered with appropriate Frontiers in Neurology | www.frontiersin.org 9 June 2020 | Volume 11 | Article 541 Leermakers et al. Adenosine Nucleotides Control ClC-1 Function A B C D E F G FIGURE 4 | Glycolysis-inhibition resulted in a repetitive action potential firing-induced ClC-1-dependent rise in Gm in human muscle fibers. (A–C) Representative traces of the membrane potential recorded with E2 (for schematic depiction of electrodes see Figure 2A) of a control (CTRL) (A), 100 µM iodoacetate (IAA) subjected (B), and 100 µM IAA + 100 µM 9-AC + 10 nM Tetrodotoxin subjected (C) human HAM muscle fiber during the 1st, 50th, and ∼100th action potential (AP) train of the stimulation protocol have been depicted. The protocol, with a total duration of 1400 ms, consisted of two square hyperpolarizing currents used for determination of Gm (−30 nA, 50 ms duration) and a train of large amplitude depolarizing current pulses to trigger a short train of action potentials [10 pulses (300 nA, 30 Hz, 1 ms)]. (D–F) Gm of CTRL (D), IAA subjected (E), and IAA/9-AC subjected (F) individual HAM muscle fiber are depicted over time. (G) The percentage of HAM muscle fibers that showed an increase in Gm that was >2000 µS/cm2 during the first 200 s of action potential stimulation. Fisher’s exact tests corrected for multiple comparisons were performed to test significance of observed differences between CTRL (n = 3), IAA (n = 9), and IAA/9-AC (n = 12), and p < 0.05* is depicted. Frontiers in Neurology | www.frontiersin.org 10 June 2020 | Volume 11 | Article 541 Leermakers et al. Adenosine Nucleotides Control ClC-1 Function A B FIGURE 5 | Rise in Gm is accelerated in murine McArdle EDL muscle fibers compared with wild-type EDL muscle fibers. (A) Murine EDL muscle fibers of McArdle (gray lines) and genetic wild-type (black line) mice were subjected repetitive action potential stimulation and Gm of is depicted over time. (B) Time until 50% of Gm(max) is reached was extracted from sigmoid-fitted data and depicted. Fibers were subjected to 200x the stimulation protocol [1500 ms, 15 pulses (300 nA, 30 Hz, 1 ms)], 100x the recovery protocol [1500 ms, 0 pulses], and to a re-stimulation period that consisted out of 100x the stimulation protocol. Gray vertical bars represent the recovery period during the stimulation protocol. Kruskal-Wallis and Dunn’s post-hoc test was performed to test significance of observed differences between wild-type (n = 9), McArdle #1 (n = 9), and McArdle #2 (n = 13), and p < 0.05* is depicted. reservations, and more experiments are required to explore this fibers from McArdle mice, that have an impaired intra-muscular in further detail. glucose homeostasis, exhibit accelerated increase of membrane conductance during repetitive action potential firing. Together, these results suggest that ClC-1 function is regulated by the DISCUSSION level of adenosine nucleotides, and that the channel operates as a sensor of the metabolic state in skeletal muscle fibers. ClC-1 The metabolic requirement of skeletal muscle fibers increases thereby represents a mechanistic link between metabolism and many-fold during physical activity. Although the muscular ATP muscle fiber excitability that can be envisioned to have a role replenishing capacity is extensive, a fiber type-specific exhaustion in muscle fatigue during exercise and, possibly, neuromuscular of the metabolic state occurs during intense exercise. Indeed, disease including McArdle disease. [ATP] was reported to decline by 80% in fast-twitch and 25% in slow-twitch human muscle fibers after 25 s of maximal Whole Cell Patch Clamp of Muscle Fibers exercise. These changes in [ATP] were generally accompanied by corresponding increases in inositol monophosphate (IMP) Show Increased ClC-1 Opening Probability levels, showing that the exercise led marked decline in the Under Conditions of Low Intracellular ATP adenosine nucleotides pool in the active muscle fibers (21). The Levels amplitude of this decrease suggest that severe energy depletion Previous patch clamp studies using heterologous expressed ClC- is a physiologically relevant event during high-intensity activity, 1 reported that adenosine nucleotides cause a pH dependent especially for fast-twitch muscle fibers. inhibition of ClC-1 (17, 36, 37). Others were, however, unable The physiological importance of a decline in adenosine to reproduce these findings (38). This apparent discrepancy nucleotides and altered metabolic profile for muscle function between studies was later explained by the high sensitivity of has been studied extensively and several cellular mechanisms the ATP-ClC-1 binding to the redox state of the cell (39). involved in the excitation-contraction coupling are sensitive to Although voltage clamping of mammalian muscle fibers has the specific metabolites. A correlation between metabolic state already been used to measure chloride currents for over 25 years of muscle fibers and their performance is evident, suggesting (40), the adenosine nucleotide sensitivity of ClC-1 in native that metabolically driven fatigue mechanisms exist in skeletal mammalian muscle fibers has not been explored. We now show muscle. The current study shows that depletion of adenosine that ATP-depletion results in a hyperpolarizing shift of the ClC-1 nucleotides results in an increased ClC-1 open probability in open probability in native skeletal muscle fibers, confirming the skeletal muscle, and that the inhibition of glycolysis results in an expression system-obtained data from previous studies in a more accelerated increase in membrane conductance during repetitive physiological relevant setting (17, 36). action potential firing in both slow- and fast-twitch muscle The whole cell patch clamping used in the current study does fiber types. Interestingly, this accelerated increase of membrane have some experimental limitations that must be considered. conductance was present in both rodent and human muscle As muscle fibers have a complex cellular geometry (size and fibers, and it was found to be ClC-1 dependent. Moreover, as composition), complete voltage control during the experiments a proof-of-principle, this study indicates that fast-twitch muscle may have been compromised. At least it must be assumed that Frontiers in Neurology | www.frontiersin.org 11 June 2020 | Volume 11 | Article 541 Leermakers et al. Adenosine Nucleotides Control ClC-1 Function the T-tubular membrane was not fully under voltage control due of low membrane conductance during onset of action potential to the T-tubular lumen imposing substantial resistance to current was previously shown to be caused by protein kinase C- flow (16). Furthermore, it is unlikely that the level of ATP was mediated ClC-1 inhibition, and has been associated with well- fully controlled by the pipette solution due to the large fiber preserved muscle fiber excitability (15). In our experiments, the volume (41). The change in the ClC-1 activation with 0 mM ATP low membrane conductance and well-preserved excitability was in the pipette was nevertheless of a clear magnitude, and the maintained for the complete protocol of action potential firing in limitations considered here indicate that our results may be an the untreated human muscle fibers. When similar experiments underestimation of the effect of ATP on ClC-1. were conducted in the presence of iodoacetate, a marked and The ATP depletion-induced shift in activation of ClC-1 results consistent rise in membrane conductance was observed during in a higher ClC-1 opening at resting membrane potential in the repeated action potential firing in the human muscle fibers. native muscle fibers and, predictably, would cause an increase This rise in membrane conductance led to complete loss of in the resting membrane conductance of resting skeletal muscle muscle fiber excitability in many fibers and, importantly, this fibers. Adenosine nucleotide mediated regulation of ClC-1 could activity-induced rise in membrane conductance was significantly therefore provide a mechanistic link between metabolic state of abolished in the presence of a ClC-1 inhibitor. Although these muscle fibers and their excitability. This was next explored in findings show that ClC-1 has the capacity to activate during muscle fibers during repetitive firing of action potentials. metabolic compromised conditions in human muscle fibers, it remains to be established if ClC-1 also activates under control Effect of Pharmacological Inhibition of conditions if stimulations are either intensified or extended Glycolysis on Regulation of Resting beyond 2000 action potentials in these fibers. It is possible that such ClC-1 activation during prolonged muscle activity would Membrane Conductance During Repetitive occur in a subset of human fibers most closely comparable to the Action Potential Firing in Muscle Fibers of EDL muscle fibers of the rat. Rat and Human Taken together, the findings from these two series of Previous studies showed that repetitive firing of action potentials experiments show that the rise in membrane conductance during results in the activation of both ClC-1 and KATP ion channels, repeated firing of action potentials is a conserved capacity which markedly increase the resting membrane conductance between different muscle fibers and species. In the currently leading to compromised muscle fiber excitability (22). This explored rat slow-twitch and human muscle fibers, the rise increase in membrane conductance has thus far only been in conductance was only observed with glycolytic inhibition. observed in fast-twitch muscle fibers and not in slow-twitch Although this is an indication that these muscle fibers were fibers. This fiber-type specific activation of ClC-1 and KATP not metabolically challenged by the imposed activity, and ion channels is compatible with fast-twitch fibers undergoing that the adenosine nucleotide levels were well-preserved in the largest depression in adenosine nucleotides during intense these muscle fibers during the activity, especially for human muscle activity. To further explore the role of the metabolic abdominal muscle fibers longer stimulation periods are needed state for the activity-induced rise in membrane conductance, two to verify this. Although we did not study intra-muscular series of experiments were conducted using both rat and human adenosine nucleotide concentrations in the current study, it is muscles to confirm translatability from rodent to man. known from literature that 2DG and iodoacetate both result First, the effect of pharmacological inhibition of glycolysis in depletion of adenosine nucleotides in challenged cardiac with 2DG on the regulation of the resting membrane muscle (42). Nevertheless, it would be interesting to study if the conductance during repeated firing of action potentials was rise in membrane conductance starts at similar intra-muscular explored in rat fast- and slow-twitch muscle fibers. With adenosine nucleotide concentrations in glycolysis-inhibited and glycolysis inhibition, the rise in membrane conductance control fibers and possibly make inter-species comparisons. happened rapidly after onset of action potential firing in both fast- and slow-twitch muscle fibers. This rise in membrane conductance was observed significantly later in untreated Role of ClC-1 in the Rise in Membrane fast-twitch fibers, and it was not observed at all in untreated Conductance During Repeated Action slow-twitch fibers. This shows that the ion channels that trigger Potential Firing the increased membrane conductance during prolonged action Collectively, the patch clamping data and the observations potential firing have the capacity to be activated in both types in human muscle during repeated firing of action potentials of muscle fibers. The absence of the increase in membrane suggest that the ClC-1-mediated chloride conductance is largely conductance in slow-twitch fibers under control conditions responsible for the accelerated rise in membrane conductance is likely due to the relatively stable metabolic state in active during prolonged muscle activity. The primary data implicating slow-twitch fibers, where adenosine nucleotide levels might not ClC-1 channels in the rise in membrane conductance during decrease to a level that triggers ClC-1 activation. muscle activity stems, in the present experiments, from the Second, when similar experiments were conducted in observation from human muscle fibers where pharmacological untreated abdominal rectus muscle fibers from human, the blockage of ClC-1 resulted in the abolishment of the rise in onset of muscle activity caused a reduction in the membrane membrane conductance. This data is in line with previous data, conductance as has been reported previously (15). This state showing that the rise in membrane conductance during repetitive Frontiers in Neurology | www.frontiersin.org 12 June 2020 | Volume 11 | Article 541 Leermakers et al. Adenosine Nucleotides Control ClC-1 Function action potential firing in rat fast-twitch muscle was to a large remained polarized throughout the experimental protocol, and extend mediated by ClC-1 activation (22). Although we clearly the membrane conductance of all fibers returned quickly to show an important role for ClC-1 for the rise in membrane baseline during the recovery period, all fibers were considered conductance during prolonged action potential firing, a possible healthy and viable and, thus, the data to be reliable. The currently role for KATP should not be disregarded. Since both ClC-1 and presented McArdle data only served as proof-of-concept, to KATP channels are sensitive to the metabolic state of muscle verify the pharmacological glycolysis inhibition data in a non- fibers, these channels may be speculated to introduce some pharmacological genetic system. Together with the data from degree of redundancy in the capacity of muscle fibers to shut pharmacological glycolysis inhibition, these data converge to a down muscle excitability in the case of challenged metabolic conclusion that the observed acceleration of the rise in membrane state (22). conductance is physiologically relevant and a conserved response Although the membrane conductance is extremely important to low energy status. Due to the limited amount of observations for functional excitation-contraction coupling, multiple cellular that we gathered from McArdle mice, substantial precautions mechanisms that regulate down-stream sarcoplasmic reticulum must be taken when extrapolating and interpreting the current Ca2+ release are sensitive to the metabolic state of muscle fibers. data the McArdle disease per se. Indeed, previous studies not only showed that the sarcoplasmic Combined, our data clearly indicates the importance reticulum Ca2+ release was greatly impaired by both low of a functional energy homeostasis for proper membrane [ATP] and by the inhibition of glycolysis with 2DG or IAA, conductance physiology and the data shows that ClC-1 acts as but they also showed that the opening of the skeletal muscle a metabolic sensor that increases membrane conductance and Ca2+ release channel Ryanodine-receptor 1 (RyR1) itself is limits muscle excitability when energy status is low. dependent on ATP-binding (43, 44). Interestingly, both ClC- 1 and RyR1 seem to have similar sensitivities to [ATP], where DATA AVAILABILITY STATEMENT a decline below 1 mM ATP causes activation or inhibition, respectively. Therefore, it is possible that adenosine nucleotides The datasets generated for this study are available on request to have a universal role as regulators of the excitation contraction the corresponding author. coupling and that coordinated shutdown of muscle activation by several cellular mechanisms can take place if [ATP] declines ETHICS STATEMENT below 1 mM. The animal study was reviewed and approved by Danish Effect of Genetically Compromised Animal Experiments Inspectorate. The studies involving Glycolysis on Regulation of the Resting human participants were reviewed and approved by Danish Membrane Conductance During Repetitive Ethics Committee, Region Midtjylland, Comité I. The Firing of Action Potentials—Role in patients/participants provided their written informed consent to participate in this study. Neuromuscular Disease Although the currently used McArdle mice were generated to model human McArdle disease (28), they were included AUTHOR CONTRIBUTIONS in this manuscript as a, proof-of-concept, genetic model PL, FP, TP, JV, TK, and THP contributed conception and design for impaired muscle glucose and energy homeostasis. These of the study. PL, KD, KH, and AR contributed to acquisition mice were characterized by complete absence of the glycogen of data. PL, KH, AR, TK, and THP contributed to analysis or phosphorylase (a.k.a. myophosphorylase), and in a previous interpretation of data. PL and THP contributed to writing the study they were shown to have a dramatically decreased time first draft of the manuscript. KH and AR wrote sections of the to fatigue (28). Moreover, the currently included EDL muscle manuscript. All authors contributed to manuscript revision, read of McArdle mice showed clear signs of structural damage, and and approved the submitted version. displayed impaired twitch and tetanic force production (45). The currently presented data, that shows that the membrane conductance rises after around 1500 action potentials in FUNDING McArdle wild-type (i.e., C57BL/6 PYGMwt/wt ) muscle fibers, corresponds exactly to reliable membrane conductance data from This work was kindly funded by Aarhus University Research an extensive previous characterization of C57BL/6 EDL fiber Foundation (AUFF) NOVA. cable parameters (23). From the two different McArdle knock- in mice (PYGMR50X/R50X ) tested here, only one showed a clear ACKNOWLEDGMENTS accelerated rise in membrane conductance. Although it is unclear why this response was observed in only one of the mice, it We would like to thank Vibeke Uhre and Dorte Olsson from is possible that not all animals were affected equally by the Aarhus university for the scientific and technical support in the mutation. As all the reported data was obtained in fibers that presented work. Frontiers in Neurology | www.frontiersin.org 13 June 2020 | Volume 11 | Article 541 Leermakers et al. Adenosine Nucleotides Control ClC-1 Function REFERENCES 22. Pedersen TH, de Paoli FV, Flatman JA, Nielsen OB. Regulation of ClC-1 and KATP channels in action potential-firing fast-twitch muscle fibers. J Gen 1. Westerblad H, Bruton JD, Katz A. Skeletal muscle: energy metabolism, Physiol. (2009) 134:309–22. doi: 10.1085/jgp.200910290 fiber types, fatigue and adaptability. Exp Cell Res. (2010) 316:3093–9. 23. Riisager A, Duehmke R, Nielsen OB, Huang CL, Pedersen TH. Determination doi: 10.1016/j.yexcr.2010.05.019 of cable parameters in skeletal muscle fibres during repetitive firing of action 2. Allen DG, Lamb GD, Westerblad H. Skeletal muscle potentials. J Physiol. (2014) 592:4417–29. doi: 10.1113/jphysiol.2014.280529 fatigue: cellular mechanisms. Physiol Rev. (2008) 88:287–332. 24. Smith IC, Gittings W, Huang J, McMillan EM, Quadrilatero J, Tupling doi: 10.1152/physrev.00015.2007 AR, et al. Potentiation in mouse lumbrical muscle without myosin light 3. Mc AB. Myopathy due to a defect in muscle glycogen breakdown. Clin Sci. chain phosphorylation: is resting calcium responsible? J Gen Physiol. (2013) (1951) 10:13–35. 141:297–308. doi: 10.1085/jgp.201210918 4. Wan JJ, Qin Z, Wang PY, Sun Y, Liu X. Muscle fatigue: general understanding 25. Edstrom L, Hultman E, Sahlin K, Sjoholm H. The contents of high-energy and treatment. Exp Mol Med. (2017) 49:e384. doi: 10.1038/emm. phosphates in different fibre types in skeletal muscles from rat, guinea- 2017.194 pig and man. J Physiol. (1982) 332:47–58. doi: 10.1113/jphysiol.1982.sp 5. Fahlke C, Rosenbohm A, Mitrovic N, George AL Jr, Rudel R. Mechanism 014399 of voltage-dependent gating in skeletal muscle chloride channels. Biophys J. 26. Banas K, Clow C, Jasmin BJ, Renaud JM. The KATP channel Kir6.2 (1996) 71:695–706. doi: 10.1016/S0006-3495(96)79269-X subunit content is higher in glycolytic than oxidative skeletal muscle 6. Jentsch TJ, Steinmeyer K, Schwarz G. Primary structure of torpedo marmorata fibers. Am J Physiol Regul Integr Comp Physiol. (2011) 301:R916–25. chloride channel isolated by expression cloning in xenopus oocytes. Nature. doi: 10.1152/ajpregu.00663.2010 (1990) 348:510–4. doi: 10.1038/348510a0 27. Real-Martinez A, Brull A, Huerta J, Tarraso G, Lucia A, Martin MA, et al. 7. Koch MC, Steinmeyer K, Lorenz C, Ricker K, Wolf F, Otto M, Low survival rate and muscle fiber-dependent aging effects in the McArdle et al. The skeletal muscle chloride channel in dominant and recessive disease mouse model. Sci Rep. (2019) 9:5116. doi: 10.1038/s41598-019- human myotonia. Science. (1992) 257:797–800. doi: 10.1126/science.13 41414-8 79744 28. Nogales-Gadea G, Pinos T, Lucia A, Arenas J, Camara Y, Brull A, et al. 8. Pusch M, Steinmeyer K, Jentsch TJ. Low single channel conductance of the Knock-in mice for the R50X mutation in the PYGM gene present with major skeletal muscle chloride channel, ClC-1. Biophys J. (1994) 66:149–52. McArdle disease. Brain. (2012) 135(Pt 7):2048–57. doi: 10.1093/brain/ doi: 10.1016/S0006-3495(94)80753-2 aws141 9. Palade PT, Barchi RL. Characteristics of the chloride conductance in 29. Skov M, De Paoli FV, Lausten J, Nielsen OB, Pedersen TH. Extracellular muscle fibers of the rat diaphragm. J Gen Physiol. (1977) 69:325–42. magnesium and calcium reduce myotonia in isolated ClC-1 chloride doi: 10.1085/jgp.69.3.325 channel-inhibited human muscle. Muscle Nerve. (2015) 51:65–71. 10. Kwiecinski H, Lehmann-Horn F, Rudel R. The resting membrane parameters doi: 10.1002/mus.24260 of human intercostal muscle at low, normal, and high extracellular potassium. 30. Haggmark T, Thorstensson A. Fibre types in human abdominal muscles. Muscle Nerve. (1984) 7:60–5. doi: 10.1002/mus.880070110 Acta Physiol Scand. (1979) 107:319–25. doi: 10.1111/j.1748-1716.1979.tb 11. Bryant SH, Morales-Aguilera A. Chloride conductance in normal and 06482.x myotonic muscle fibres and the action of monocarboxylic aromatic acids. J 31. Lueck JD, Mankodi A, Swanson MS, Thornton CA, Dirksen RT. Muscle Physiol. (1971) 219:367–83. doi: 10.1113/jphysiol.1971.sp009667 chloride channel dysfunction in two mouse models of myotonic dystrophy. 12. Rudel R, Senges J. Mammalian skeletal muscle: reduced chloride conductance J Gen Physiol. (2007) 129:79–94. doi: 10.1085/jgp.200609635 in drug-induced myotonia and induction of myotonia by low-chloride 32. Cheung A, Dantzig JA, Hollingworth S, Baylor SM, Goldman YE, Mitchison solution. N-S Arch Pharmacol. (1972) 274:337–47. doi: 10.1007/BF00501271 TJ, et al. A small-molecule inhibitor of skeletal muscle myosin II. Nat Cell Biol. 13. Hutter OF, Noble D. The chloride conductance of frog skeletal muscle. J (2002) 4:83–8. doi: 10.1038/ncb734 physiol. (1960) 151:89–102. 33. Zhang SJ, Andersson DC, Sandstrom ME, Westerblad H, Katz A. 14. de Paoli FV, Broch-Lips M, Pedersen TH, Nielsen OB. Relationship between Cross bridges account for only 20% of total ATP consumption during membrane Cl- conductance and contractile endurance in isolated rat muscles. submaximal isometric contraction in mouse fast-twitch skeletal muscle. J Physiol. (2013) 591:531–45. doi: 10.1113/jphysiol.2012.243246 Am J Physiol Cell Physiol. (2006) 291:C147–54. doi: 10.1152/ajpcell.005 15. Riisager A, de Paoli FV, Yu WP, Pedersen TH, Chen TY, Nielsen OB. 78.2005 Protein kinase C-dependent regulation of ClC-1 channels in active human 34. Hodgkin AL, Rushton WA. The electrical constants of a crustacean nerve muscle and its effect on fast and slow gating. J Physiol. (2016) 594:3391–406. fibre. Proc R Soc Med. (1946) 134:444–79. doi: 10.1098/rspb.1946.0024 doi: 10.1113/JP271556 35. Albuquerque EX, Thesleff S. A comparative study of membrane 16. Pedersen TH, Huang LH, Fraser JA. An analysis of the relationships between properties of innervated and chronically denervated fast and slow subthreshold electrical properties and excitability in skeletal muscle. J Gen skeletal muscles of the rat. Acta Physiol Scand. (1968) 73:471–80. Physiol. (2011) 138:73–93. doi: 10.1085/jgp.201010510 doi: 10.1111/j.1365-201X.1968.tb10886.x 17. Bennetts B, Rychkov GY, Ng HL, Morton CJ, Stapleton D, Parker 36. Bennetts B, Parker MW, Cromer BA. Inhibition of skeletal muscle ClC-1 MW, et al. Cytoplasmic ATP-sensing domains regulate gating of skeletal chloride channels by low intracellular pH and ATP. J Biol Chem. (2007) muscle ClC-1 chloride channels. J Biol Chem. (2005) 280:32452–8. 282:32780–91. doi: 10.1074/jbc.M703259200 doi: 10.1074/jbc.M502890200 37. Tseng PY, Bennetts B, Chen TY. Cytoplasmic ATP inhibition of 18. Tseng PY, Yu WP, Liu HY, Zhang XD, Zou X, Chen TY. Binding of ATP to CLC-1 is enhanced by low pH. J Gen Physiol. (2007) 130:217–21. the CBS domains in the C-terminal region of CLC-1. J Gen Physiol. (2011) doi: 10.1085/jgp.200709817 137:357–68. doi: 10.1085/jgp.201010495 38. Zifarelli G, Pusch M. The muscle chloride channel ClC-1 is not 19. Meyer S, Savaresi S, Forster IC, Dutzler R. Nucleotide recognition by the directly regulated by intracellular ATP. J Gen Physiol. (2008) 131:109–16. cytoplasmic domain of the human chloride transporter ClC-5. Nat Struct Mol doi: 10.1085/jgp.200709899 Biol. (2007) 14:60–7. doi: 10.1038/nsmb1188 39. Zhang XD, Tseng PY, Chen TY. ATP inhibition of CLC-1 is controlled 20. Pedersen TH, Macdonald WA, de Paoli FV, Gurung IS, Nielsen OB. by oxidation and reduction. J Gen Physiol. (2008) 132:421–8. Comparison of regulated passive membrane conductance in action potential- doi: 10.1085/jgp.200810023 firing fast- and slow-twitch muscle. J Gen Physiol. (2009) 134:323–37. 40. Fahlke C, Rudel R. Chloride currents across the membrane of doi: 10.1085/jgp.200910291 mammalian skeletal muscle fibres. J Physiol. (1995) 484(Pt 2):355–68. 21. Karatzaferi C, de Haan A, Ferguson RA, van Mechelen W, Sargeant AJ. doi: 10.1113/jphysiol.1995.sp020670 Phosphocreatine and ATP content in human single muscle fibres before 41. Pusch M, Neher E. Rates of diffusional exchange between small cells and after maximum dynamic exercise. Pflugers Arch. (2001) 442:467–74. and a measuring patch pipette. Pflugers Arch. (1988) 411:204–11. doi: 10.1007/s004240100552 doi: 10.1007/BF00582316 Frontiers in Neurology | www.frontiersin.org 14 June 2020 | Volume 11 | Article 541 Leermakers et al. Adenosine Nucleotides Control ClC-1 Function 42. Pirolo JS, Allen DG. Assessment of techniques for preventing glycolysis in Conflict of Interest: The authors declare that the research was conducted in the cardiac muscle. Cardiovasc Res. (1986) 20:837–44. doi: 10.1093/cvr/20.11.837 absence of any commercial or financial relationships that could be construed as a 43. Kockskamper J, Zima AV, Blatter LA. Modulation of sarcoplasmic reticulum potential conflict of interest. Ca2+ release by glycolysis in cat atrial myocytes. J Physiol. (2005) 564(Pt 3):697–714. doi: 10.1113/jphysiol.2004.078782 Copyright © 2020 Leermakers, Dybdahl, Husted, Riisager, de Paoli, Pinós, Vissing, 44. Dutka TL, Lamb GD. Effect of low cytoplasmic [ATP] on excitation- Krag and Pedersen. This is an open-access article distributed under the terms contraction coupling in fast-twitch muscle fibres of the rat. J Physiol. (2004) of the Creative Commons Attribution License (CC BY). The use, distribution or 560(Pt 2):451–68. doi: 10.1113/jphysiol.2004.069112 reproduction in other forums is permitted, provided the original author(s) and the 45. Krag TO, Pinos T, Nielsen TL, Brull A, Andreu AL, Vissing J. Differential copyright owner(s) are credited and that the original publication in this journal muscle involvement in mice and humans affected by McArdle disease. J is cited, in accordance with accepted academic practice. No use, distribution or Neuropathol Exp Neurol. (2016) 75:441–54. doi: 10.1093/jnen/nlw018 reproduction is permitted which does not comply with these terms. Frontiers in Neurology | www.frontiersin.org 15 June 2020 | Volume 11 | Article 541