The role of action potential changes in depolarization-induced failure of excitation contraction coupling in mouse skeletal muscle

Excitation-contraction coupling (ECC) is the process by which electrical excitation of muscle is converted into force generation. Depolarization of skeletal muscle resting potential contributes to failure of ECC in diseases such as periodic paralysis, intensive care unit acquired weakness and possibly fatigue of muscle during vigorous exercise. When extracellular K+ is raised to depolarize the resting potential, failure of ECC occurs suddenly, over a narrow range of resting potentials. Intracellular recordings of action potentials (APs) in individual mouse skeletal muscle fibers during depolarization of the resting potential revealed small APs are still generated at resting potentials at which force production has failed. Simultaneous imaging of Ca2+ transients and recording of APs demonstrated failure to generate Ca2+ transients when APs peaked at potentials more negative than -30 mV. An AP property that closely correlated with failure of the Ca2+ transient was the integral of AP voltage with respect to time. Simultaneous recording of Ca2+ transients and APs with electrodes separated by 1.6 mm revealed AP conduction fails when APs peak below -21 mV. We hypothesize propagation of APs and generation of Ca2+ transients are governed by distinct AP properties: AP conduction is governed by AP peak, whereas Ca2+ release from the sarcoplasmic reticulum is governed by AP integral of voltage with respect to time. The reason distinct AP properties may govern separate steps of ECC is the kinetics of the ion channels involved in the different steps of ECC. Na channels, which govern propagation, have rapid kinetics such that propagation is insensitive to AP width (and integral) whereas Ca2+ release is governed by movement of gating charges in Cav1.1 channels, which have slower kinetics such that Ca2+ release is sensitive to AP width (and integral). The quantitative relationships established between resting potential, AP properties, AP conduction and Ca2+ transients provide the foundation for future studies of failure of ECC induced by depolarization of the resting potential.Data set #1: Force data for Fig 1A. It consists of 1 sheet. Column A lists the time during the experiment. Columns B through M represent 1 muscle each and show the normalized force generated at each time point. The title states the extracellular K concentration that was infused. Column N lists when the various extracellular K concentration was changed. Data set #2: Intracellular recording of APs and ΔF/F for Fig 1. The excel file consists of 5 sheets. The extracellular K concentration the muscle was incubated in is the title of each sheet. Each sheet has 5 columns with data in them. The first column in each sheet is the date of the experiment. The second column lists the muscle fiber number from each muscle. Each muscle was used for several different K concentrations such that it appears on more than 1 data sheet. For this reason, the fiber number sometimes starts at a large number as the muscle was already used for a lower K concentration. The third column is the resting potential at the time the action potential was recorded. The fourth column of data is the action potential peak, and the fifth column of data is the normalized ΔF/F triggered by the action potential. The ΔF/F in the data sheet is normalized to 100%. Data sets #3, 4 and 5: These data sets were used to generate the plots in Fig 2 E, F and G respectively. Each excel file has data from the same 12 fibers and if desired could be combined into 1 excel spread sheet with all the values for each fiber. In each file, data from each fiber is represented as 2 columns. The first is the x axis for each plot and the second column is the y axis for each plot. The rows represent measurements taken every 5s during infusion of solution containing 16 mM K. Data set #6: This data set has the data used to perform the analysis shown in Fig 3. There are 6 rows of data for each fiber. The first row is the resting potentials, the second is the AP peak, the third is the normalized AP peak, the fourth is the normalized delta F/F, the fifth is the AP area and the sixth is the normalized AP area. The columns in red starting at column BU were normalizations that were not correct. The values were used to calculate the correct normalization. Data set #7 is the data used to generate the plots for the two example fibers shown in Fig 4 where the electrodes are 1.6 mm apart. There are two data sheets. Each sheet represents the analysis for 1 of the fibers. Column A is the date the recording was performed. Column B is the fiber number. Column C is the sweep number; each sweep is separated by 5s in time. Column D is the action potential peak for each sweep recorded 1.6 mm from the stimulating electrode. Column E is the resting potential. Columns F through Y are the un-normalized image intensities of the 20 ROIs analyzed for the signal generated following stimulation for each sweep. Column F is the ROI by the stimulating electrode and column Y is by the recording electrode. Data set #8 is the data used to generate the plots for the two example fibers shown in Fig 5 where the stimulating and recording electrodes are close together. There are two data sheets. Each sheet represents the analysis for 1 of the fibers. Column A is the date the recording was performed. Column B is the fiber number. Column C is the sweep number; each sweep is separated by 5s in time. Column D is the action potential peak for each sweep. Column E is the resting potential. Columns F through Y are the un-normalized image intensities of the 20 ROIs analyzed for the signal generated following stimulation for each sweep. The stimulating and recording electrodes are both near the ROI at column O. Data set #9 is the data used to generate the plots for the fiber shown in Fig 6 where the stimulating and recording electrodes are close together. There is one data sheet. Column A is the date the recording was performed. Column B is the fiber number. Column C is the sweep number; each sweep is separated by 5s in time. Column D is the action potential peak for each sweep. Column E is the resting potential. Columns F through Y are the un-normalized image intensities of the 20 ROIs analyzed for the signal generated following stimulation for each sweep. The stimulating and recording electrodes are both near the ROI at column O. At row 57 the stimulus duration was increased from 0.2 ms to 5 ms such that "action potential" peak was increased from -35 to +56 mV. Funding provided by: National Institute of Arthritis and Musculoskeletal and Skin DiseasesCrossref Funder Registry ID: http://dx.doi.org/10.13039/100000069Award Number: AR074985Funding provided by: Muscular Dystrophy AssociationCrossref Funder Registry ID: http://dx.doi.org/10.13039/100005202Award Number: 602459Two electrode intracelllular recordings from mouse EDL fibers and muscle force recordings from mouse EDL muscles. Simultaneous with the intracellular recording, Ca transients were imaged. Raw traces have been analyzed and data entered into excel spreadsheets