Crucial role of sodium channel fast inactivation in muscle fibre inexcitability in a rat model of critical illness myopathy

Abstract

Critical illness myopathy is an acquired disorder in which skeletal muscle becomes electrically inexcitable. We previously demonstrated that inactivation of Na+ channels contributes to inexcitability of affected fibres in an animal model of critical illness myopathy in which denervated rat skeletal muscle is treated with corticosteroids (steroid denervated; SD). Our previous work, however, did not address the relative importance of membrane depolarization versus a shift in the voltage dependence of fast inactivation in causing inexcitability. It also remained unknown whether changes in the voltage dependence of activation or slow inactivation play a role in inexcitability. In the current study we found that a hyperpolarizing shift in the voltage dependence of fast inactivation of Na+ channels is the principal factor underlying inexcitability in SD fibres. Although depolarization tends to decrease excitability, it is insufficient to account for inexcitability in SD fibres since many normal and denervated fibres retain normal excitability when depolarized to the same resting potentials as affected SD fibres. Changes in the voltage dependence of activation and slow inactivation of Na+ channels were also observed in SD fibres; however, the changes appear to increase rather than decrease excitability. These results highlight the importance of the change in fast inactivation in causing inexcitability of SD fibres.

Figure 1. Failure of action potentials in control muscle depolarized with high external potassium

A-D, traces from control fibres depolarized with increasing external K⁺ concentrations. Each panel shows superimposed traces of action potentials following increasing current pulses. In A the resting potential was −87 mV; in B, C and D the resting potential had been depolarized to −65, −63 and −61 mV, respectively. A, five superimposed traces are shown, two of which are suprathreshold. With increasing current strength the action potential occurs earlier, but the peak potential of the action potential is unchanged. B, action potentials are still all or none, but the peak amplitude is decreased and threshold for action potential initiation occurs at a more depolarized potential. Despite the decrease in excitability such fibres were classified as excitable. In C, however, the action potential is no longer all or nothing. With increased current pulses the peak of the action potential increases (arrow). Fibres with graded actions potential amplitudes such as the one shown were classified as indeterminate. D, no action potential can be elicited despite current pulses that depolarized the fibre to close to −15 mV. Such fibres were classified as inexcitable.

Figure 2. Resting potential versus excitability in control, SD and denervated muscle

In each graph, the percentages of excitable, indeterminate and inexcitable fibres are shown at resting potentials ranging from −75 to −58 mV. Excitable fibres that can generate an action potential peaking at a potential greater than +10 mV are shown in black; indeterminate fibres that generate an action potential peaking at potentials of −5 to +10 mV are dark grey; inexcitable fibres with no action potential or action potentials peaking at less than −10 mV are light grey. In the top graph excitability is plotted for control muscle depolarized to various resting potentials with high K⁺. The middle graph plots excitability for SD muscle and the bottom graph plots excitability for denervated muscle. The number of fibres in each resting potential (mV) group is (control, SD, denervated): −75 to −71 (3, 8, 2); −70, −69 (10, 12, 5); −68, −67 (9, 9, 3); −66, −65 (15, 8, 5); −64, −63 (22, 10, 8); −62, −61 (16, 16, 6); −60 to −58 (8, 14, 7).

Figure 3. The average voltage dependence of fast inactivation in control, denervated and SD muscle fibres

The normalized Na⁺ current evoked by a step to −20 mV is plotted versus the 50 ms prepulse potential for control, denervated and SD fibres. Both denervated and SD fibres have been categorized into 3 groups based on excitability (excitable, indeterminate and inexcitable). All control fibres are excitable. The inactivation curves for control, denervated excitable and SD excitable are very nearly superimposed and have the most positive voltage dependence. The inactivation curves for indeterminate denervated and SD fibres are shifted towards more hyperpolarized potentials relative to the curves for control and excitable fibres. The curves for denervated and SD indeterminate fibres are nearly superimposed in the voltage range of the resting potentials of these fibres (−60 to −70 mV). The inactivation curves for inexcitable denervated and SD fibres have the most hyperpolarized voltage dependence and are nearly identical. For all curves except for denervated inexcitable, error bars represent the standard error of the mean. No error bars are shown for denervated inexcitable fibres since only 2 such fibres were studied. The 3 curves for SD fibres are represented by open squares and continuous lines, the 3 curves for denervated fibres are represented by open circles and dashed-dotted lines, and the control curve is represented by open triangles and a dotted line.

Figure 4. The average voltage dependence of slow inactivation in control and SD muscle

The normalized Na⁺ current evoked by a step to −20 mV is plotted versus the holding potential for control and SD fibres. There is little difference in the membrane potential at which the normalized Na⁺ current is half-maximal between the 4 groups. However, there is a steeper voltage dependence of slow inactivation in control and SD excitable fibres relative to inexcitable and SD indeterminate fibres (Table 2). Because of the shallower voltage dependence of slow inactivation, inexcitable and SD indeterminate fibres have less slow inactivation at membrane potentials more positive than −80 mV. Error bars represent the s.e.m..

Figure 5. Determination of the voltage dependence of fast, slow and total inactivation in a control muscle fibre

The curve representing fast inactivation is plotted using circles and the curve representing slow inactivation is plotted using squares. Both the fast and slow inactivation curves are plotted using continuous lines. When the percentage of Na⁺ channels inactivated from fast and slow inactivation is multiplied, the resultant curve represents total Na⁺ channel inactivation (dashed line). The 3 curves obtained can then be used to determine the percentage of Na⁺ channels inactivated at the resting potential (−86 mV in the fibre shown, ↑) or at −64 mV (↓).

Figure 6. The average voltage dependence of Na⁺ channel activation in control and SD muscle

A, superimposed current traces evoked by stepping from −130 mV to voltages of −70 to −10 mV in 10 mV increments in a control fibre. The steps to −70 and −60 mV do not elicit an inward Na⁺ current. Steps from −50 to −20 mV elicit inward currents that increase in amplitude. With the final step to −10 mV, the inward current begins to decrease. Following the inward Na⁺ current there is an outward K⁺ current. The amplitude of this outward current continues to increase as the voltage steps become more positive. B, the normalized I–V relationship is plotted versus membrane potential for control and 3 categories of SD fibres. SD inexcitable fibres have inward currents that peak on average at membrane potentials between −30 and −40 mV. Peak current amplitude in indeterminate fibres occurred at −30 mV while SD excitable and control fibres had inward Na⁺ currents that peaked at −20 mV. C, the normalized Na⁺ conductance calculated from the data in B assuming a reversal potential of +45 mV is plotted versus membrane potential. The midpoint of activation of SD inexcitable fibres is shifted towards more negative voltages by almost 15 mV relative to control and SD excitable fibres (P < 0.01; Table 1). Error bars represent the s.e.m.

Figure 7. Correlations between Na⁺ channel gating properties

A-C, plots of correlations between activation, fast inactivation and slow inactivation in SD and control muscle fibres. A, the correlation between more positive voltage dependencies of activation and fast inactivation is shown (R= 0.72, P < 0.01). SD inexcitable and indeterminate fibres tend to have both more negative midpoints of both activation and fast inactivation than do SD excitable and control fibres. B, the correlation between a more positive voltage dependence of fast inactivation and a steeper voltage dependence of slow inactivation is shown (R=−0.61, P < 0.01). C, the correlation between a more positive voltage dependence of activation and a steeper voltage dependence of slow inactivation is shown (R=−0.74, P < 0.01). All fits are linear. More points are present in A than in B and C since fibres were included in which the voltage dependence of slow inactivation was not measured. ▪, SD inexcitable fibres; ▵, SD indeterminate fibres; ○, SD excitable fibres; ♦, control fibres.