The Role of Action Potential Waveform in Failure of Excitation Contraction Coupling

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, ICU 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 range of several mV of resting potential. While some studies have hypothesized the sudden failure of ECC is due to all-or-none failure of excitation, other studies suggest failure of excitation is graded. Intracellular recordings of action potentials (APs) in individual fibers during depolarization revealed that APs do not fail in an all-or-none manner. Simultaneous imaging of Ca2+ transients during depolarization revealed failure over a narrow range of resting potentials. An AP property that closely correlated with the sudden failure of the Ca2+ transient was the integral of AP voltage with respect to time. We hypothesize the close correlation is due to the combined dependence on time and voltage of Ca2+ release from the sarcoplasmic reticulum. The quantitative relationships established between resting potential, APs and Ca2+ transients provide the foundation for future studies of depolarization-induced failure of ECC in diseases such as periodic paralysis.


Abstract:
28 Excitation contraction coupling (ECC) is the process by which electrical excitation of muscle is 29 converted into force generation. Depolarization of skeletal muscle resting potential contributes 30 to failure of ECC in diseases such as periodic paralysis, ICU acquired weakness and possibly 31 fatigue of muscle during vigorous exercise. When extracellular K + is raised to depolarize the 32 resting potential, failure of ECC occurs suddenly, over a range of several mV of resting potential. 33 While some studies have hypothesized the sudden failure of ECC is due to all-or-none failure of  Introduction: 46 The process by which electrical excitation of muscle is converted into force generation is 47 known as excitation contraction coupling (ECC). Successful ECC involves invasion of action 48 potentials into a network of membrane invaginations in muscle known as the transverse tubules 49 (t-tubules) (Adrian et al., 1969). Depolarization in the t-tubules activates Cav1.1 channels, 50 which triggers opening of ryanodine receptors, Ca 2+ exit from the sarcoplasmic reticulum and 51 force production (Melzer et al., 1995;Dulhunty, 2006;Bannister and Beam, 2013;Hernandez-52 Ochoa and Schneider, 2018).

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While the idea of all-or-none muscle contraction dates back more than 100 years (Pratt,66 1917), the mechanism underlying the near all-or none failure of force generation in the setting of 67 depolarization of the resting potential remains unknown. One hypothesis is that the sudden 68 failure of ECC is due to all-or-none failure of excitability (Renaud and Light, 1992; Cairns et al.,

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Two studies have suggested reduction in AP peak can cause reduction in force generation 77 (Cairns et al., 2003;Gong et al., 2003). These studies are consistent with graded failure of force 78 production in individual fibers.

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To determine the mechanism underlying the sudden failure of ECC, we measured muscle 80 force, APs and Ca 2+ transients in muscle in which the resting potential was depolarized by 81 elevation of extracellular K + . In agreement with previous studies, mouse EDL twitch force 82 decreased dramatically over a narrow range of extracellular K + concentrations (Cairns et al.,     and 5% CO 2 . Solutions containing elevated concentrations of KCl (3.5, 10, 12, 14, and 16 mM) 110 and with corresponding reduction in NaCl (118, 111.5, 109.5, and 105.5 mM respectively) to 111 maintain a constant osmolarity were used to induce depolarization. The EDL was stimulated with 112 two electrodes placed perpendicularly to the muscle in the bath. The force transducer was 113 controlled by a 305C two-channel controller (Aurora Scientific) and digitized by a Digidata 114 1550B digitizer (Molecular Devices). A S-900 pulse generator (Dagan) was used to generate 0.1 115 ms 5V twitch stimulations to the muscle. The pulse generator was triggered using pCLAMP 11 116 data acquisition and analysis software. The optimal length was determined by adjusting the 117 tension of the muscle until maximal twitch force was achieved. During force recordings, the 118 muscle was exposed to normal K + solution for 20 minutes, followed by high K + solution (10 mM 119 to 16mM) for 45 minutes, and then washed again with normal K + solution for 25 minutes to 120 follow recovery. The EDL was stimulated with a twitch pulse every 5 minutes, and force was 121 recorded.

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To prevent contraction, muscles were loaded with 50μM BTS (N-benzyl-p-  where Out represents the dependent variable (either AP peak or Ca 2+ image intensity), V is the 157 independent voltage variable (either resting potential or AP peak), LV is the limiting value when 158 V is very low (toward more negative), HV is the limiting value when V is very high (toward more 159 positive), V50 is the value of V at which Out is halfway between HV and LV, and k is the slope 160 factor. All voltages and the variable k are expressed in mV, and Ca 2+ image intensity is in 161 arbitrary units between 1 for maximum intensity for each experiment and 0.

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Data for recordings from different muscles were analyzed using nested analysis of 165 variance with n as the number of mice, with data presented as mean ± SD. Comparisons of 166 different parameters recorded from the same fiber were compared using the paired students t-test.

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Following the initial increase in force, there was a decline that became faster with higher 179 levels of extracellular K + (Fig 1A). With return to solution containing normal K + , force 180 recovered rapidly. Force generated 40 minutes following infusion of high K + was steeply 181 dependent on extracellular K + , such that force was near normal in 10 mM K + but near 0 in 14 and 182 16 mM K + . The mean resting potential 20-40 minutes after infusion of each concentration of K + 183 was measured in a separate set of experiments (n = 80 fibers from 4 muscles for each K + 184 concentration) and those data were used to construct a plot of force versus mean resting 185 potential. There was a steep loss of force over a narrow range of resting potentials: normal force 186 was generated at a mean resting potential of -65.3 ± 1.9 mV in 10 mM K + and almost no force 187 was generated at a mean resting potential of -59.3 ± 1.9 mV in 14 mM K + (Fig 1B). Our finding 188 is similar to a previous report of steep dependence of force production on resting potential 189 (Cairns et al., 1997).

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The sudden loss of force with depolarization of muscle has been hypothesized to be due APs peaked at 30.2 ± 2.7 mV. With depolarization of the resting potential to -65.3 ± 1.9 mV, the 203 peak was reduced to -4.4 ± 3.9 mV and at a resting potential of -55.4 ± 2.0 mV, the peak 204 averaged -36.5 ± 8.0 mV (Fig 1D). Reduction of the mean AP peak from 30.2 mV to -4.4 mV 205 was associated with little to no reduction in force, whereas reduction of the peak from -4 mV to -206 36 mV was associated with almost complete loss of force (Fig 1 E). These data raise the 207 possibility that there may be a threshold for AP peak above which APs trigger full contraction 208 and below which there is failure of ECC. membrane potential (Fig 2 A, B), which was accompanied by up to 15 mV of reduction in AP 230 peak (Fig 2 B). Infusion of a solution containing 16 mM K + causes substantial depolarization of 231 the resting membrane potential and reduction in the AP peak beyond that seen with impalement 232 alone (Fig 2 C, D). Consistent with recordings taken from populations of fibers, infusion of 16 233 mM K + caused graded reduction of the AP peak from a maximum ranging from +15 to +35 mV . For these fits, the HV 238 limit, which represents the minimal AP peak when resting potential was elevated, was 239 constrained to be between -30 mV and -50 mV. The V50 for the resting potential at which AP 240 peak was half maximal was -58.2 ± 3.3 mV, the slope factor k was 1.8 ± 0.6 mV, and the average 241 value of the half-maximal AP peak at the V50 value was -15.5 ± 4.9 mV (n =12 fibers from 6 242 mice).  image intensity when resting potential was -70 mV, was fixed to 1, and the HV limit was 281 constrained to be between 0 and 0.1. The resting potential at which Ca 2+ transient was half 282 maximal was -57.5 ± 3.4 mV with a slope factor of 0.4 ± 0.2 mV, which was significantly 283 steeper than the slope for reduction in AP peak (p < 1 x 10 -5 , paired t-test, n =12 fibers from 6 284 mice, Fig 3E). 285 We plotted the reduction in Ca 2+ transient versus AP peak (Fig 3F), and fit the data with a The finding that all-or none-failure of the AP was not the mechanism underlying normalized AP amplitude and the Ca 2+ transient. A loss of 11.6 ± 1.7 mV of resting potential 317 was required to reduce AP amplitude from 90% to 10% of maximum (Fig 4D). In contrast, the 318 Ca 2+ transient was reduced from 90% to 10% of maximum with a loss of only 4.1 ± 2.4 mV of 319 resting potential (Fig 4D, p < 1x10 -6 vs APs, n=12, paired student's t-test). This statistically 320 significant difference led us to look for another AP parameter that decreased more sharply with 321 depolarization of the resting potential.

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As shown in Fig 3F, an AP peak above -30 mV is required to consistently trigger Ca 2+ 323 release. We thus set AP peaks of -30 mV or below to 0 and normalized AP amplitude. At 324 mildly depolarized resting potentials, drops in normalized AP peaks were accompanied by 325 increases in the Ca 2+ transient (Fig 4A, B, Fig 3E)  in Ca 2+ transient (Fig 4A, B). When the normalized Ca 2+ transient was plotted against the 328 normalized AP peak, the mean R 2 was 0.65 ± 0.14 ( Fig 4C, D, n =12 fibers). This was not the 329 close relationship we were hoping to find. 330 We next considered whether changes in AP kinetics might affect the Ca 2+ transient. It depolarization of the resting potential (Fig 4A, B). When the normalized Ca 2+ transient was 342 plotted against normalized AP area, the mean R 2 value was 0.86 ± 0.11 (Fig 4C, D and provide a conduit for release of Ca 2+ from the sarcoplasmic reticulum into the myoplasm.

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The total gating charge is dependent on both voltage and time (Schneider and Chandler, 1973). also non-linear. The use of the AP area metric as described in this work is therefore a 417 simplification of the underlying processes.

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There are several limitations of our study. One is that the Ca 2+ indicator used is relatively  However, with depolarization of the resting potential there is graded reduction of the AP peak 440 such that AP amplitude ranges from 120 mV to below 10 mV (the current study and (Rich and