Ryanodine receptor modulation by caffeine challenge modifies Na+ current properties in intact murine skeletal muscle fibres

Figures

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  Figure 1. (A) Plan view of the muscle preparation after being pinned down at both ends in the bath. The soleus muscle (in grey) is overlaid by gastrocnemius. The shaded box in the centre represents the region of gastrocnemius fibres used for patch clamping. (B) Loose patch clamp experiment: Muscle preparation pinned down in KH solution. Pipette mounted at 45° to allow 90° contact of the bent tip with the fibre surface. The pipette is connected to a suction syringe13. (C) Equivalent circuit of loose patch clamp electrode on muscle membrane. Solution in the pipette clamped at Vpip. The voltage error arising from the flow of currents through the patch resistance (Rpatch) and the cell resistance (Rcell), along with the pipette resistance (Rpip) corrected mainly using a bridge circuit in the loose patch clamp amplifier13. (D) Complete set of pulse protocols carried out on a patch being investigated. This double test pulse protocol first superimposed a −40 mV hyperpolarising pre-pulse, lasting 50 ms, on the resting potential, RMP. This was followed by a depolarising V1 pulse varying in amplitude to test voltages between RMP and (RMP + 80) mV in +5 mV increments. This was followed by a V2 pulse…

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  Figure 2. Results of simple pulse protocols to examine INa at early times following caffeine challenge using two experimental procedures. Following obtaining control records prior to caffeine challenge (A–D) panel (i), in (A), the patch electrode was withdrawn prior to introduction of 0.5 mM caffeine, and then restored prior to imposition of test steps (ii) in the following 1–3 min. In (B) the patch seal was maintained before, during and following introduction of 0.5 mM caffeine through imposition of the test steps (ii). The latter procedure was repeated following pipette withdrawal and restoration of the patch seal (iii). (C,D) adopted identical respective procedures as (A,B) using a 2 mM caffeine concentration.

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  Figure 3. Families of normalized current traces obtained from the double pulse protocol in response to caffeine challenge at a concentration of 0.5 mM in the absence (A,B) or presence (C,D) of 10 μM dantrolene. (A,C) Control traces obtained in KH solution, prior to caffeine challenge in the absence (A) or presence of dantrolene (C). (B,D) Families of records obtained at successive (i) 7–8 min, (ii) 20–21 min, (iii) 35–37 min and (iv) 45–46 min intervals after addition of 0.5 mM caffeine-containing KH solution into the bath in the absence (B) or presence of dantrolene (D).

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  Figure 4. Activation (left panels) and inactivation I-V curves (right panels of each pair) (mean of I1 or I2 ± standard error of mean (SEM) against each V1 excursion) in response to caffeine challenge at a concentration of 0.5 mM in the absence (A) or the presence (B) of 10 μM dantrolene. Experiments first obtained families of INa records before addition of caffeine in (A(i)), (B(i)). Families of INa records then obtained in intervals (ii) 1–10 minutes, (iii) 11–25 minutes (iv) 26–40 minutes and (v) 41–55 min intervals after adding 0.5 mM caffeine.

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  Figure 5. Activation and inactivation I-V curves (mean of I1 or I2 ±standard error of mean (SEM) against each V1 excursion) for the (i) control condition prior to addition of caffeine, and (ii), 11–25 minutes and (iii) 26–40 minutes after addition of 0.5 mM caffeine using an expanded range of V1 in the absence of dantrolene.

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  Figure 6. Alterations in (i) Imax, (ii) V* and (iii) k derived from current-voltage (I-V) activation and inactivation curves followed from successive patches studied at different times before (t < 0 min) and following addition of 0.5 mM caffeine in the absence (A) and presence (B) of 10 μM dantrolene.

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  Figure 7. Families of normalized current traces obtained from the double pulse protocol in response to caffeine challenge at a concentration of 2 mM in the absence (A,B) or the presence (C,D) of 10 μM dantrolene. (A,C) Control traces obtained in KH solution, prior to caffeine challenge. (B,D) Families of records obtained at successive (i) 3 or 5 min, (ii) 19 or 21 min and (iii) 29 or 31 min after addition of 2 mM caffeine-containing KH solution into the bath in (B) the absence or (D) presence of dantrolene.

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  Figure 8. Activation (left panels) and inactivation I-V curves (right panels) (mean of I1 or I2 ± standard error of mean (SEM)) against each V1 excursion before (i) and following (ii) introduction of 2 mM caffeine in the absence (A) or the presence (B) of 10 μM dantrolene. Families of INa records were obtained 2–15 min (ii), 16–25 min (iii), and 26–40 min (iv) after adding 2 mM caffeine.

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  Figure 9. Alterations in (i) Imax, (ii) V* and (iii) k derived from current-voltage (I-V) activation (left panels) and inactivation (right panels) followed from successive patches studied at different times before (t < 0 min) and following addition of 2 mM caffeine in the absence (A) and presence (B) of 10 μM dantrolene.

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  Figure 10. Correlation between observed ryanodine receptor (RyR) agonist and antagonist actions and (A) Feedforward Nav1.5 mediated depolarisation triggering DHPR-RyR coupling causing SR Ca2+ release that is (B) inhibited by RyR-antagonist dantrolene, and previous studies reporting: (C,D) Challenge by (C) 0.5 mM caffeine increasing RyR-mediated Ca2+ release and FKBP12 dissociation reducing Nav1.4 availability thereby modifying Nav1.4 properties and (D) 2 mM caffeine, where Ca2+ release causes sustained RyR inactivation with net SR Ca2+ uptake, reduced FKBP12 dissociation, and contrasting Nav1.4 effects. Blue arrows: fluxes; green and red dotted arrows: excitatory and inhibitory actions.