A novel signalling pathway originating in mitochondria modulates rat skeletal muscle membrane excitability

Abstract

Single skeletal muscle fibres from rat and cane toad were mechanically skinned and stimulated either electrically by initiating action potentials in the sealed transverse (t-) tubular system or by ion substitution causing depolarisation of the t-system to pre-determined levels. Depression of mitochondrial ATP-producing function with three diverse mitochondrial function antagonists (azide: 1-10 mM; oligomycin 1 microg ml-1 and carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP) 1 microM), under conditions in which the cytosolic ATP was maintained high and constant, invariably reduced the excitability of rat fibres but had no obvious effect on the excitability of toad fibres, where mitochondria are less abundant and differently located. The reduction in excitability linked to mitochondria in rat fibres appears to be caused by depolarisation of the sealed t-system membrane. These observations suggest that mitochondria can regulate the functional state of mammalian muscle cells and have important implications for understanding how the balance between ATP utilisation and ATP production is regulated at the cellular level in general and in mammalian skeletal muscle fibres in particular.

Figure 1. Effect of mitochondrial antagonists on the excitability of mechanically skinned rat EDL fibres

A, schematic representation of mammalian fibre ultrastructure and of the mechanical skinning procedure. Note that paired long mitochondria are transversely located at the I-band level wrapped around the contractile apparatus and in contact with SR but clearly separated from t-tubules. B, representative force responses to electrical stimulation, exposure to low-Mg²⁺ solution and to a maximally Ca²⁺-activating solution (pCa 4.5) from one preparation equilibrated in standard K-HDTA solutions with and without mitochondrial antagonists. C, summary of the twitch-force response data (n = 6–11). D, summary of the force responses when replacing the K-HDTA solution with a Na-HDTA solution (n = 3–5). Z, Z-line; A, A-band; I, I-band. Here and in subsequent figures: Con, control; Oligo, 1 μg ml⁻¹ oligomycin; FCCP, 1 μm FCCP.

Figure 2. Effect of mitochondrial antagonists on the excitability of mechanically skinned cane toad muscle fibres

A, representative force responses from one preparation equilibrated in standard K-HDTA solutions with and without mitochondrial antagonists. Summary of twitch data (B) (n = 6–7) and of force responses when replacing the K-HDTA solution with a Na-HDTA solution (C) (n = 7, 5 and 2 for azide, oligomycin and FCCP, respectively).

Figure 3. Effects of t-system depolarisation on force responses in rat fibres

A, summary of twitch-force responses of partly depolarised rat fibres incubated in the presence of 43 mm K⁺ (V_m about −60 mV) (n = 7, 6, 4 and 3 for controls, 1 mm azide, 1 μg ml⁻¹ oligomycin and 1 μm FCCP, respectively). B, representative twitch responses at high temporal resolution in the same fibre when partially depolarised in the 43 mm K⁺ solution and when equilibrated in standard myoplasmic solution with 1 mm azide or 1 μg ml⁻¹ oligomycin. Responses were normalised to the same peak height and overlaid. C, action potential-induced twitch-force responses (continuous line) and force responses induced by t-system depolarisation with the Na-HDTA solution (dotted line) in rat fibres equilibrated in solutions of different myoplasmic [K⁺]. The lower x-axis is the estimated t-system membrane potential (see text). The test twitch responses were bracketed by twitch responses in the standard K-HDTA solution. Data points were best fitted by the Bolzmann-equation (F_relative = 1/(1 + exp(F₅₀ – [K⁺])/slope)), with half-maximal responses (F₅₀) at 50 and 27 mm K⁺ and slopes of 12 and 9 mm for the twitch and ion substitution curves, respectively.

Figure 4. Change in rat fibre excitability after rapidly stopping the Na⁺-K⁺-ATPase by Na⁺ removal from the myoplasmic environment

Twitch-force responses from a representative fibre elicited every 3 s in a 0 Na-solution in the absence and the presence of 1 mm azide. The last trace is the normalised response of the change in the level of activation of the contractile apparatus when changing from a Na-EGTA (pCa 6.0) to a K-EGTA (pCa 6.0) solution. This change in level of activation is due to the lower Ca²⁺ sensitivity in the presence of Na⁺ (Fink et al. 1986) and approximates the time taken (2–3 s) to remove Na⁺ from the myoplasmic environment.