Lower Ca2+ enhances the K+-induced force depression in normal and HyperKPP mouse muscles

Figures

• 

  Figure 1.. Lowering [Ca2+]e enhanced the K+-induced force depression in wild type and HyperKPP soleus and EDL, the effects being greater in HyperKPP. After 45 min at 4.7 mM K+ and 2.4, 1.3, or 0.3 mM Ca2+, [K+]e was increased to 10 mM. All muscles were later exposed to 5 µM salbutamol (SAL; protocol 1 in Materials and methods). (A, C, E, and G) Examples of tetanic force traces. (B, D, F, and H) Changes in mean tetanic force over time. To clearly show the Ca2+ effects on the K+-induced force depression, mean tetanic force is expressed as a percent of the force measured at the [Ca2+]e just before the increase in [K+]e. Vertical bars represent the SE for five or six muscles.

• 

  Figure 2.. Lowering [Ca2+]e significantly shifted the tetanic force-[K+]e relationship toward lower [K+]e, the effect being greater for HyperKPP than wild type soleus and EDL. Changes in [K+]e and [Ca2+]e were as described for protocol 1. To clearly show the Ca2+ effects on the K+-induced force depression, mean tetanic force is expressed as a percentage of the force measured after a 45-min exposure period to 2.4, 1.3, or 0.3 mM Ca2+; that is, just before the increase in [K+], as shown in Fig. 1. The tetanic force–[K+]e relationship (no salbutamol; A, C, E, and G) was obtained using the force measured 45 min after the increase in [K+]e (time 50 min; Fig. 1). The tetanic force–[K+]e relationship in the presence of 5 µM salbutamol (B, D, F, and H) was obtained using the force measured 10 min after salbutamol addition. For clarity, the relationships in the absence and presence of salbutamol were separated. The dashed horizontal line indicates 100%. Vertical bars represent the SE of five to nine muscles. *, Force–[K+]e relationship was significantly shifted to lower [K+]e compared with 2.4 mM Ca2+. †, Mean tetanic force of HyperKPP muscles was significantly different from that of wild type. ¥,…

• 

  Figure 3.. Increasing [K+]e significantly enhanced the tetanic force depression at low [Ca2+], the effect being greater for HyperKPP than wild type soleus and EDL. Changes in [K+]e and [Ca2+]e were as described for protocol 2. To clearly show how K+ affects the Ca2+–tetanic force relationship, mean tetanic force is expressed as a percent of the force measured after a 45-min exposure to 4.7, 8, or 12 mM K+; that is, just before the decrease in [Ca2+]e. Vertical bars represent the SE of three to five muscles. §, Mean tetanic force significantly different from 100%. *, Mean tetanic force significantly different from that at 4.7 mM K+. †, Mean tetanic force of HyperKPP muscles significantly different that of wild type (same [K+]e and [Ca2+]e). ANOVA and LSD, P < 0.05.

• 

  Figure 4.. Lowering [Ca2+]e reduced the capacity of wild type and HyperKPP EDL to maintain a plateau phase at elevated [K+]e. Fade represents the force drop after peak force had been reached, was calculated as the difference in force between peak force and the force at 200 ms (end of the stimulation period), and was expressed as a percentage of the peak force. Changes in [K+]e and [Ca2+]e were according to protocol 1. The control fade–[K+]e relationship (i.e., no salbutamol [SAL]) was obtained using the force measured 45 min after the increase in [K+]e (time 50 min; Fig. 1). The fade–[K+]e relationship in the presence of 5 µM salbutamol was obtained using the force measured 10 min after the salbutamol addition. Vertical bars represent the SE of five to nine muscles. §, [K+]e at which mean fade became significantly greater than at 4.7 mM K+. *, Mean fade was significantly different from that at 2.4 mM Ca2+. ¥, Significant effect of salbutamol. ANOVA and LSD, P < 0.05.

• 

  Figure 5.. Examples of force increase between elicited contractions in HyperKPP soleus when [K+]e was increased from 4.7 to 6 mM while [Ca2+]e was 0.3 mM. (A) Example of a slow increase in unstimulated force being in this case small and late after the increase in [K+]e. (B) Example of a fast and transient contracture (see arrow) followed by a large and slow increase in unstimulated force. Notice the decrease in unstimulated force following the addition of 5 µM salbutamol. The chart recorder trace was generated by averaging three data points every second for the baseline and drawing a vertical line from the minimum to the maximum force when a contraction was elicited.

• 

  Figure 6.. Lowering [Ca2+]e increased the occurrence frequency of unstimulated force and contractures. Occurrence frequency of unstimulated force and contractures in soleus (A) and EDL (B). Numbers at the top of the bars represent the range of [K+]e (in mM) that we observed an unstimulated force/contracture and the number of muscles that generated unstimulated force/contractures over the total number of muscles tested. Mean unstimulated force of soleus (C) and EDL (D). Unstimulated force was calculated from muscles that generated it and is expressed as a percentage of the tetanic force measured at 4.7 mM K+ and 2.4 mM Ca2+. Data from different [K+]e were pooled, because the magnitude of the unstimulated forces were not significant. *, Frequency of unstimulated force or contractures (A and B) was significantly different from that at 2.4 mM Ca2+. §, Mean unstimulated force was significantly different from that at time 0 min (C and D). †, Mean unstimulated force of HyperKPP muscles was significantly different that of wild type (C and D). ANOVA and LSD, P < 0.05.

• 

  Figure 7.. Lowering [Ca2+]e significantly enhanced the K+-induced depression of membrane excitability to a greater extent in HyperKPP than in wild type soleus. (A) Examples of action potential traces generated by excitable fibers upon stimulation. Resting EM: −80 mV (i), −61 mV (ii), −54 mV (iii), −52 mV (iv); the last trace being an example of an unexcitable fiber that failed to generate an action potential. The little square change in EM preceding action potential was the stimulation artifact. (B) Mean fiber excitability expressed as a percentage of the number of fibers tested. Changes in [K+]e and [Ca2+]e were as described in protocol 3. Data for salbutamol (SAL) were divided into early measurements (first 10 min, E) and late measurements (15–30 min, L). The reversibility of the K+ and Ca2+ effects was tested by returning to the initial conditions (i.e., 4.7 mM K+ and 2.4 mM Ca2+). Vertical bars represent the SE of 71–144 fibers/5–6 muscles for high [K+]e, 277 fibers/26 muscles for wild type and 273 fibers/18 muscles for HyperKPP at 4.7 mM K+ and 2.4 mM. §, At similar [Ca2+]e, mean excitability significantly different from that at 4.7 mM K+ measured at the beginning of an experiment…

• 

  Figure 8.. Lowering [Ca2+]e significantly enhanced the K+-induced membrane depolarization in both wild type and HyperKPP soleus. Mean resting EM values were calculated from all tested fibers (mean resting EM of unexcitable and excitable fibers are respectively given in Tables 1 and 2). All [K+]e and [Ca2+]e conditions are as described in Fig. 7. Data for salbutamol are divided into early measurements (first 10 min, E) and late measurements (15–30 min, L). The reversibility of the K+ and Ca2+ effects was tested by returning to the initial conditions (i.e., 4.7 mM K+ and 2.4 mM Ca2+). Vertical bars represent the SE of 71–144 fibers/5–6 muscles for high [K+]e, 277 fibers/26 muscles for wild type and 273 fibers/18 muscles for HyperKPP at 4.7 mM K+ and 2.4 mM. §, At similar [Ca2+]e, mean resting EM significantly different from that at 4.7 mM K+ measured at the beginning of an experiment (i.e., in A). *, At similar [K+]e, mean resting EM at 0.3 mM Ca2+ significantly different from that at 2.4 mM. †, At the same [K+]e and [Ca2+]e, mean resting EM of HyperKPP fibers significantly different that of wild type fibers. ¥, Significant effect of salbutamol. ANOVA and LSD, P < 0.05.

• 

  Figure 9.. Lowering [Ca2+]e significantly increased the number of wild type soleus fibers exposed to 10 mM K+ and HyperKPP fibers exposed to both 4.7 and 10 mM K+ with resting EM less negative than the mean resting EM of unexcitable fibers. Resting EM values were divided in bin of 5 mV, and the number of fibers in each bin is expressed as a percentage of the total number of fibers tested. Mean resting EM of unexcitable wild type and HyperKPP fibers was −62 and −57 mV, respectively (Table 1), and is represented by a vertical dark yellow line. *, Frequency distribution at 0.3 mM Ca2+ significantly shifted compared with 2.4 mM Ca2+. Fisher test, P < 0.05.

• 

  Figure 10.. Salbutamol (SAL; 5 µM) at 10 mM K+ significantly shifted the frequency distribution of resting EM toward more negative potential only in HyperKPP solei. Resting EM values were divided in bin of 5 mV, and the number of fibers in each bin is expressed as a percent of the total number of fibers tested. Mean resting EM of unexcitable wild type and HyperKPP fibers was −62 and −57 mV, respectively (Table 1), and is represented by a vertical purple line. ¥, Salbutamol significantly shifted the frequency distribution of resting EM. Fisher test, P < 0.05.

• 

  Figure 11.. Lowering [Ca2+]e significantly enhanced the K+-induced overshoot depression in both wild type and HyperKPP soleus. All [K+]e and [Ca2+]e conditions are as described in Fig. 7. Data in the presence of salbutamol were divided into early measurements (first 10 min, E) and late measurements (15–30 min, L). The reversibility of the K+ and Ca2+ effects was tested by returning to the initial conditions (i.e., 4.7 mM K+ and 2.4 mM Ca2+). Vertical bars represent the SE of 71–144 fibers/5–6 muscles for high [K+]e, 277 fibers/26 muscles for wild type and 273 fibers/18 muscles for HyperKPP at 4.7 mM K+ and 2.4 mM. §, At similar [Ca2+]e, mean overshoot significantly different from that at 4.7 mM K+ measured at the beginning of an experiment (i.e., in A). *, At similar [K+]e, mean overshoot at 0.3 mM Ca2+ significantly different from that at 2.4 mM. †, At the same [K+]e and [Ca2+]e, mean overshoot of HyperKPP fibers significantly different that of wild type fibers. ¥, Significant effect of salbutamol. ANOVA and LSD, P < 0.05. No AP, no action potential.

• 

  Figure 12.. At 4.7 mM K+, replacing Ca2+ by Mg2+ significantly improved mean tetanic force of HyperKPP soleus and EDL, while at 10 mM K+, it improved force for wild type soleus and HyperKPP EDL. Changes in [K+]e and [Ca2+]e were as described in protocol 1, except that the 0.3 mM Ca2+ solution also contained 5.2 mM Mg2+. (A and B) Tetanic force measured at 4.7 mM K+ and 0.3 mM Ca2+ with either 3.1 or 5.2 mM Mg2+ and expressed as a percentage of the tetanic force at 4.7 mM K+ and 2.4 mM Ca2+. (C and D) Tetanic force measured at 10 mM K+ and 0.3 mM Ca2+ and expressed as a percentage of the force measured just before [K+]e was increased (i.e., 4.7 mM and 0.3 mM Ca2+). Horizontal dash black lines indicate the position of the mean values at 2.4 mM Ca2+. Vertical bars represent the SE of five muscles. ‡, Mean tetanic force at 5.2 mM Mg2+ was significantly different from that at 3.1 mM. *, Mean tetanic force at 0.3 mM Ca2+ was significantly different from that at 2.4 mM, ANOVA and LSD, P < 0.05.