Paxilline Prevents the Onset of Myotonic Stiffness in Pharmacologically Induced Myotonia: A Preclinical Investigation

Figures

• 

  Figure 1. Scheme of a typical twitch recording with the corresponding parameters used for analysis. The red trace represents a typical recording of a myotonic muscle. The electrical stimulus has a duration of 1 ms and a voltage of 25 V. Tpeak is a marker for the steepness of the upstroke of a single twitch. The relaxation times were measured as T1/2 (ms) – time to peak half and T90/10 (ms) – time from 90 to 10%.

• 

  Figure 2. Effect of Paxilline and NS1608 on T90/10 in 9-AC induced low chloride conductance (gCl−) myotonia in murine muscle. T90/10 was recorded on six bundles of soleus (SOL; A) and extensor digitorum longus (EDL; B) isolated muscle after application of the BK channel blocker Paxilline (20 μM) or the BK channel activator NS1608 (20 μM). Paxilline caused a decrease in T90/10 in anthracene-9-carboxylic acid (9-AC; 100 μM) myotonic muscle whereas NS1608 had opposite effects. Paxilline in WT muscle caused a slight but not significant increase of relaxation time. Data displayed the T90/10 from the first 15 twitches in WT and 9-AC myotonic muscles. +Significant difference vs. 9-AC.

• 

  Figure 3. Effect of Paxilline and NS1608 in 9-AC induced low gCl− myotonia in human samples. Data were obtained from the first twitch contraction in Musculus vastus lateralis obtained from healthy donors (n = 8). Low gCl− myotonia was pharmacologically induced by 100 μM 9-AC in 4.5 mM [K+]0 KR. The BK+ channel blocker Paxilline (n = 4) or the BK+ channel opener NS 1608 (n = 4) were applied to 9-AC myotonic muscle at the concentration of 20 μM. Force (A) and T90/10 (B) were shown. Neither Paxilline nor NS1608 caused significant effects of relaxation times or force. Paxilline significantly reduced relaxation times T90/10 in 9-AC pretreated human muscle, while NS1608 significantly prolonged relaxation times in 9-AC pretreated human muscle. *Significant difference vs. 9-AC.

• 

  Figure 4. Effects of pharmacological induced myotonia by 9-AC in BK−/− mice on T90/10. The effects of BK channels were evaluated in WT and BK−/− muscle after induction of myotonia by 9-AC (B) or after application of control solution (A) on five bundles of EDL. Consecutively, WT and BK−/− muscle were tested after induction of myotonia by 9-AC (n = 7; D) or after application of control solution (n = 5; C) on SOL muscle. Data displayed the T90/10 from the first 20 twitches in WT and BK−/− after pharmacologically induced myotonia. *Significantly different vs. WT.

• 

  Figure 5. Effects of pharmacological induced myotonia by 9-AC in BK−/− mice on T1/2. Half-time of relaxation (T1/2) for single twitches in EDL (A,B) and SOL (C,D) muscles from WT and BK−/− mice in the presence of 9-AC (B,D). Figures (A,C) represent T1/2 after application of control solution. Figures (B,D) represent T1/2 after induction of myotonia by 100 μM 9-AC. *Significantly different vs. WT.

• 

  Figure 6. T-tubular co-localization of BK channels with dihydropyridine receptor (DHPR) and ryanodine receptor (RyR) SOL and EDL muscles were double immunofluorescence-labeled using a specific antibody against the BK channel (green) and the RyR type 1 (red) or DHPR (red). No staining for BK channels was observed in muscles from BK−/− mice.