DOI: 10.5061/dryad.5mkkwh7dx
title: Reduced K+ build-up in t-tubules contributes to resistance of the diaphragm to myotonia

description: Patients with myotonia congenita suffer from slowed muscle relaxation
 caused by hyperexcitability. The diaphragm is only mildly affected in
 myotonia congenita; discovery of the mechanism underlying its resistance
 to myotonia could identify novel therapeutic targets. Intracellular
 recordings from two mouse models of myotonia congenita revealed the
 diaphragm had less myotonia than either the EDL or the soleus muscles. A
 mechanism contributing to the resistance of the diaphragm to myotonia was
 reduced depolarisation of the interspike membrane potential during
 repetitive firing of action potentials, a process driven by the build-up
 of K+ in small invaginations of muscle membrane known as t-tubules. We
 explored differences between diaphragm and EDL that might underlie the
 reduction of K+ build-up in diaphragm t-tubules. A smaller size of
 diaphragm fibres, which promotes the diffusion of K+ out of t-tubules was
 identified as a contributor. Intracellular recording revealed slower
 repolarization of action potentials in the diaphragm suggesting reduced Kv
 conductance. Higher resting membrane conductance was identified suggesting
 increased Kir conductance. Computer simulation found reduction of Kv
 conductance had little effect on K+ build-up whereas increased Kir
 conductance lessened build-up, although the effect was modest. Our data
 and computer simulation suggest the opening of K+ channels during action
 potentials has little effect on K+ build-up whereas the opening of K+
 channels during the interspike interval slightly lessens K+ build-up. We
 conclude activation of K+ channels may lessen myotonia by opposing
 depolarisation to action potential threshold without worsening K+ build-up
 in t-tubules.
description: Intracellular and force recordings recordings were performed.
 Data was collected using a CED 1401 A to D board using Spike2 software
 (Cambridge Electronic Design Limited). No filtering was applied to the
 signal.
description: # Reduced K+ build-up in t-tubules contributes to resistance of the
 diaphragm to myotonia
 [https://doi.org/10.5061/dryad.5mkkwh7dx](https://doi.org/10.5061/dryad.5mkkwh7dx) These data are from a mouse model of myotonia congenita.   ## Description of the data and file structure For muscle force recordings we used the genetic model of myotonia congenita as we found the severity of myotonia changed over time when myotonia was triggered by block of Cl- channels with 100 µM 9-anthracenecarboxylic acid (9AC, the pharmacologic model of myotonia congenita) (Palade and Barchi 1977). The genetic mouse model of myotonia congenita used was *Clcn1adr-mto*/J (ClCadr) mice, which have a homozygous null mutation in the *Clcn1* gene (Jackson Labs cat #000939). For intracellular recordings 9AC was applied to trigger myotonia. This allowed us to avoid the need for a large breeding colony to produce the requisite number of recessive *Clcn1* mutants. Intracellular recordings were made from surface fibres, which were bathed in solution containing 9AC.  Genotyping of ClCadr mice was performed by sequencing as previously described to select heterozygous mice for breeding (Dupont, Denman et al. 2019).  Homozygous myotonic mice were identified by appearance and behavior as previously described (Novak, Norman et al. 2015).  Both male and female mice were used from 2 to 6 months of age. As mice with myotonia have difficulty climbing to reach food, symptomatic mice were supplied with moistened chow paste (Irradiated Rodent Diet; Harlan Teklad 2918) on the floor of the cage. Mice were sacrificed using CO2 inhalation followed by cervical dislocation. *Force recording*: *When the diaphragm was used, a slice of muscle no more than 1 cm wide was prepared. The EDL muscle was dissected out intact.  The proximal tendon of the EDL, soleus or the ribs attached to the diaphragm were secured to a stable rod in the dish used to record force. The distal tendon of the EDL, soleus or the central tendon of the diaphragm was attached to a force transducer via a small metal hook. Muscles were maintained and recorded from within 6 hours of sacrifice. The recording chamber was continuously perfused with Ringer solution containing (in mM): NaCl, 118; KCl, 3.5; CaCl2, 1.5; MgSO4, 0.7; NaHCO3, 26.2; NaH2PO4, 1.7; glucose, 5.5 (20-22°C) and equilibrated with 95% O2 and 5% CO2. Muscles were directly stimulated via platinum electrodes placed on either side of the muscle. To trigger myotonia, muscle was stimulated with 40 1 ms duration pulses delivered at 100 Hz.  Force was recorded using a CED 1401 A to D board using Spike2 software (Cambridge Electronic Design Limited). No filtering was applied to the signal. *Intracellular recording*: *Diaphragm and EDL muscles from the same mouse were dissected and studied in pairs in the same bath to ensure experimental conditions were identical. For most experiments the same solution used for force recording was perfused. Chloride substitution experiments were performed by substituting NaCl and KCl with their respective methyl-sulfonate salts and by replacing CaCl2 with CaNO2 as was previously done to induce myotonia (Mehrke, Brinkmeier et al. 1988).  Contraction was prevented by loading muscles with 50μM BTS dissolved in DMSO for 45 minutes prior to recording (Macdonald, Pedersen et al. 2005). Muscle fibres were impaled with 2 sharp microelectrodes filled with 3 M KCl solution containing 1 mM sulforhodamine 101 to allow for visualization. Electrode resistances were between 10 and 30 MΩ, and capacitance compensation was optimized prior to recording. Action potentials were evoked by a 0.2 ms injection of current ranging from 100 to 1000 nA. Membrane time constant was measured during a 200 ms injection of hyperpolarizing current ranging from 5 to 40 nA. Fibres with resting potentials more depolarized than –74 mV were discarded. Sampling frequency was 50 kHz with a 5 kHz low pass filter. *Drugs*: *µ* conotoxin GIIIA was purchased from Alamone Labs, Jerusalem, Israel (cat # STC-280). BTS (N-benzyl-p-toluenesulfonamide) was purchased from Tokyo Chemical Industry, Tokyo, Japan (cat# B3082). 9AC (9-anthracenecarboxylic acid) and sulforhodamine 101 were purchased from Millpore Sigma, Burlington, MA (cat# A89405 and cat# S7635).  The excel data files are organized according to the figure or table in the manuscript.  Labels have been added that match the labels in the figures or tables.   ## Sharing/Access information All data comes from experiments performed for the manuscript.  ## Code/Software Coding was performed using matlab version R2020a run the main.m file change following variables in pg_diaphragm_jphys_ss_1.m file, found in "parameter files" subdirectory: cell_radius: 23e-4 or 17e-4 P_KDr_max: 0.00045 or 0.000075          Kv permeability Kir.params(1): 1.5e-6 or 3e-6                    Kir permeability Can also adjust initial ion concentrations or other channel parameters in this file if desired. For creating plots, after running the simulation as described above: time values can be found by doing the following commands: times = fitData.prot{1}.times_to_sim; times = times - times(1); membrane voltage (sarcolemma) at time points run for voltage probe segment can be found in: outstruct.prot{1}.Vm_pred(:,1,fitData.uIpms.gen.voltage_probe_segment); t-tubule voltage values for all shells of the voltage probe segment can be found in: vtplot t-tubule potassium concentration for all shells of the voltage probe segment can be found by executing the following command: squeeze(outstruct.prot{1}.molecs_t_rsh{2}(:,plotstep,plotseg,:)); Potassium Nernst value for all t-tubule shells of the voltage probe segment can be found by executing the following command: squeeze(fitData.RTF.*log(outstruct.prot{1}.molecs_t_rsh{2}(:,plotstep,plotseg,:)./outstruct.prot{1}.molecs_i{2}(:,plotstep,plotseg)));