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Potassium dependent rescue of a myopathy with core-like structures in mouse.

Hanson MG, Wilde JJ, Moreno RL, Minic AD, Niswander L - Elife (2015)

Bottom Line: Myopathies decrease muscle functionality.Mutant muscle shows a persistent potassium leak and disrupted expression of regulators of potassium homeostasis.Inhibition of KATP channels or increasing interstitial potassium by diet or FDA-approved drugs can reverse the muscle weakness, fatigue-like physiology and pathology.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Colorado, Anschutz Medical Campus, Aurora, United States.

ABSTRACT
Myopathies decrease muscle functionality. Mutations in ryanodine receptor 1 (RyR1) are often associated with myopathies with microscopic core-like structures in the muscle fiber. In this study, we identify a mouse RyR1 model in which heterozygous animals display clinical and pathological hallmarks of myopathy with core-like structures. The RyR1 mutation decreases sensitivity to activated calcium release and myoplasmic calcium levels, subsequently affecting mitochondrial calcium and ATP production. Mutant muscle shows a persistent potassium leak and disrupted expression of regulators of potassium homeostasis. Inhibition of KATP channels or increasing interstitial potassium by diet or FDA-approved drugs can reverse the muscle weakness, fatigue-like physiology and pathology. We identify regulators of potassium homeostasis as biomarkers of disease that may reveal therapeutic targets in human patients with myopathy of central core disease (CCD). Altogether, our results suggest that amelioration of potassium leaks through potassium homeostasis mechanisms may minimize muscle damage of myopathies due to certain RyR1 mutations.

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Serum level measurements from Ryr1+/+ (black bars) and Ryr1AG/+ (grey bars) in 2- and 6-month old mice.Measurements of serum levels were performed on Ryr1+/+ (black bars) and Ryr1AG/+ (grey bars) in 2- and 6-month old mice (n = 6 samples per group per age for Ryr1+/+ and Ryr1AG/+).DOI:http://dx.doi.org/10.7554/eLife.02923.009
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fig4s1: Serum level measurements from Ryr1+/+ (black bars) and Ryr1AG/+ (grey bars) in 2- and 6-month old mice.Measurements of serum levels were performed on Ryr1+/+ (black bars) and Ryr1AG/+ (grey bars) in 2- and 6-month old mice (n = 6 samples per group per age for Ryr1+/+ and Ryr1AG/+).DOI:http://dx.doi.org/10.7554/eLife.02923.009

Mentions: The significant depletion of ATP in RyR1AG/+ muscle could consequently affect ATP-dependent mechanisms that normally act within the muscle, such as the influx of potassium to enhance membrane excitability. To determine whether potassium homeostasis is disrupted in Ryr1AG/+ muscle, we used the potassium indicator PBFI-AM (see ‘Materials and methods’) in combination with ratiometric potassium imaging of muscle fibers. Experiments using wild-type soleus in normal Ringer's solution (3 mM K+) showed that intracellular potassium concentration was 137 ± 7 mM (n = 23 fibers from 4 mice; Figure 4A,C). However, Ryr1AG/+ muscle showed lower intracellular potassium concentrations of 106 ± 8 mM (n = 29 fibers from 4 mice; p < 0.005; Figure 4B,C), a similar decline as that observed for wild-type muscles undergoing fatigue (McKenna et al., 2008). Moreover, Ryr1AG/+ muscle fibers showed a slow decrease in potassium fluorescent ratio intensity over the course of a minute suggesting an increase in K+ ion permeability (Figure 4D,E; n = 23 fibers in 4 Ryr1AG/+ mice and n = 29 fibers in 4 wild-type mice), which may be indicative of a potassium leak. We hypothesized that if Ryr1AG/+ muscles have a defect in potassium homeostasis due to an increase in K+ ion permeability, it might be possible to decrease the semi-permeability of the K+ ion with the addition of external K+. However, we did not observe a significant difference in serum K+ levels in 2-month old Ryr1AG/+ mice, but there was a 12 ± 3% increase in serum K+ in 6-month old Ryr1AG/+ mice relative to wild-type (Figure 4—figure supplement 1, n = 6 samples per group per age for Ryr1+/+ and Ryr1AG/+). The acute addition of 7 mM KCl to the muscle increased PBFI-AM fluorescence intensity, suggesting inhibition of a potassium leak, while 0 mM KCl decreased PBFI-AM fluorescence intensity (Figure 4D,E; n = 23 fibers in 4 Ryr1AG/+ mice and n = 29 fibers in 4 wild-type mice). Glibenclamide (2 μM) selectively binds to and inhibits SUR1/2 (Porat et al., 2011), subunits of the KATP6.1 and KATP6.2 channels in muscle (Pedersen et al., 2009). In wild-type adult mouse skeletal muscle, glibenclamide protects against fatigue caused by tetanic force (Duty and Allen, 1995). Additionally, in chick muscle fibers, glibenclamide increases the twitch and tetanus tension induced by application of caffeine, an agonist of RyR1 (Andrade et al., 2011). The acute addition of 2 μM glibenclamide to the 3 mM KCl bath resulted in increased intracellular K+ in wild-type (n = 17 fibers in 4 mice) and Ryr1AG/+ (n = 16 fibers in 4 mice) soleus muscle fibers (Figure 4D,F). Upon bath application of 7 mM KCl for 1.5 hr to examine a continuous counterbalancing of the K+ leak, the intracellular potassium concentration in the mutant soleus increased to 132 ± 8 mM, similar to wild-type (Figure 4G, n = 25 fibers in 4 Ryr1AG/+ mice and n = 26 fibers in 4 wild-type mice). Upon bath application of 2 μM glibenclamide for 1.5 hr, the intracellular potassium concentration in the mutant soleus increased to 138 ±12 mM, similar to wild-type (Figure 4H, n = 28 fibers in 4 Ryr1AG/+ mice and n = 28 fibers in 4 wild-type mice). The decline in intracellular K+ fluorescence in Ryr1AG/+ muscle compared to wild-type muscle (Figure 4A–E) and the increase in intracellular K+ upon KATP channel inhibition (Figure 4G,H) suggest an increase in KATP channel activity in Ryr1AG/+ muscle. These data suggest that increased extracellular K+ ion and inhibition of KATP channels might prevent the excessive K+ leak and protect the mutant muscle from decreased intracellular concentrations of K+.10.7554/eLife.02923.008Figure 4.Detection and compensation of an internal potassium leak in RyR1AG/+ muscle.


Potassium dependent rescue of a myopathy with core-like structures in mouse.

Hanson MG, Wilde JJ, Moreno RL, Minic AD, Niswander L - Elife (2015)

Serum level measurements from Ryr1+/+ (black bars) and Ryr1AG/+ (grey bars) in 2- and 6-month old mice.Measurements of serum levels were performed on Ryr1+/+ (black bars) and Ryr1AG/+ (grey bars) in 2- and 6-month old mice (n = 6 samples per group per age for Ryr1+/+ and Ryr1AG/+).DOI:http://dx.doi.org/10.7554/eLife.02923.009
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4309926&req=5

fig4s1: Serum level measurements from Ryr1+/+ (black bars) and Ryr1AG/+ (grey bars) in 2- and 6-month old mice.Measurements of serum levels were performed on Ryr1+/+ (black bars) and Ryr1AG/+ (grey bars) in 2- and 6-month old mice (n = 6 samples per group per age for Ryr1+/+ and Ryr1AG/+).DOI:http://dx.doi.org/10.7554/eLife.02923.009
Mentions: The significant depletion of ATP in RyR1AG/+ muscle could consequently affect ATP-dependent mechanisms that normally act within the muscle, such as the influx of potassium to enhance membrane excitability. To determine whether potassium homeostasis is disrupted in Ryr1AG/+ muscle, we used the potassium indicator PBFI-AM (see ‘Materials and methods’) in combination with ratiometric potassium imaging of muscle fibers. Experiments using wild-type soleus in normal Ringer's solution (3 mM K+) showed that intracellular potassium concentration was 137 ± 7 mM (n = 23 fibers from 4 mice; Figure 4A,C). However, Ryr1AG/+ muscle showed lower intracellular potassium concentrations of 106 ± 8 mM (n = 29 fibers from 4 mice; p < 0.005; Figure 4B,C), a similar decline as that observed for wild-type muscles undergoing fatigue (McKenna et al., 2008). Moreover, Ryr1AG/+ muscle fibers showed a slow decrease in potassium fluorescent ratio intensity over the course of a minute suggesting an increase in K+ ion permeability (Figure 4D,E; n = 23 fibers in 4 Ryr1AG/+ mice and n = 29 fibers in 4 wild-type mice), which may be indicative of a potassium leak. We hypothesized that if Ryr1AG/+ muscles have a defect in potassium homeostasis due to an increase in K+ ion permeability, it might be possible to decrease the semi-permeability of the K+ ion with the addition of external K+. However, we did not observe a significant difference in serum K+ levels in 2-month old Ryr1AG/+ mice, but there was a 12 ± 3% increase in serum K+ in 6-month old Ryr1AG/+ mice relative to wild-type (Figure 4—figure supplement 1, n = 6 samples per group per age for Ryr1+/+ and Ryr1AG/+). The acute addition of 7 mM KCl to the muscle increased PBFI-AM fluorescence intensity, suggesting inhibition of a potassium leak, while 0 mM KCl decreased PBFI-AM fluorescence intensity (Figure 4D,E; n = 23 fibers in 4 Ryr1AG/+ mice and n = 29 fibers in 4 wild-type mice). Glibenclamide (2 μM) selectively binds to and inhibits SUR1/2 (Porat et al., 2011), subunits of the KATP6.1 and KATP6.2 channels in muscle (Pedersen et al., 2009). In wild-type adult mouse skeletal muscle, glibenclamide protects against fatigue caused by tetanic force (Duty and Allen, 1995). Additionally, in chick muscle fibers, glibenclamide increases the twitch and tetanus tension induced by application of caffeine, an agonist of RyR1 (Andrade et al., 2011). The acute addition of 2 μM glibenclamide to the 3 mM KCl bath resulted in increased intracellular K+ in wild-type (n = 17 fibers in 4 mice) and Ryr1AG/+ (n = 16 fibers in 4 mice) soleus muscle fibers (Figure 4D,F). Upon bath application of 7 mM KCl for 1.5 hr to examine a continuous counterbalancing of the K+ leak, the intracellular potassium concentration in the mutant soleus increased to 132 ± 8 mM, similar to wild-type (Figure 4G, n = 25 fibers in 4 Ryr1AG/+ mice and n = 26 fibers in 4 wild-type mice). Upon bath application of 2 μM glibenclamide for 1.5 hr, the intracellular potassium concentration in the mutant soleus increased to 138 ±12 mM, similar to wild-type (Figure 4H, n = 28 fibers in 4 Ryr1AG/+ mice and n = 28 fibers in 4 wild-type mice). The decline in intracellular K+ fluorescence in Ryr1AG/+ muscle compared to wild-type muscle (Figure 4A–E) and the increase in intracellular K+ upon KATP channel inhibition (Figure 4G,H) suggest an increase in KATP channel activity in Ryr1AG/+ muscle. These data suggest that increased extracellular K+ ion and inhibition of KATP channels might prevent the excessive K+ leak and protect the mutant muscle from decreased intracellular concentrations of K+.10.7554/eLife.02923.008Figure 4.Detection and compensation of an internal potassium leak in RyR1AG/+ muscle.

Bottom Line: Myopathies decrease muscle functionality.Mutant muscle shows a persistent potassium leak and disrupted expression of regulators of potassium homeostasis.Inhibition of KATP channels or increasing interstitial potassium by diet or FDA-approved drugs can reverse the muscle weakness, fatigue-like physiology and pathology.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Colorado, Anschutz Medical Campus, Aurora, United States.

ABSTRACT
Myopathies decrease muscle functionality. Mutations in ryanodine receptor 1 (RyR1) are often associated with myopathies with microscopic core-like structures in the muscle fiber. In this study, we identify a mouse RyR1 model in which heterozygous animals display clinical and pathological hallmarks of myopathy with core-like structures. The RyR1 mutation decreases sensitivity to activated calcium release and myoplasmic calcium levels, subsequently affecting mitochondrial calcium and ATP production. Mutant muscle shows a persistent potassium leak and disrupted expression of regulators of potassium homeostasis. Inhibition of KATP channels or increasing interstitial potassium by diet or FDA-approved drugs can reverse the muscle weakness, fatigue-like physiology and pathology. We identify regulators of potassium homeostasis as biomarkers of disease that may reveal therapeutic targets in human patients with myopathy of central core disease (CCD). Altogether, our results suggest that amelioration of potassium leaks through potassium homeostasis mechanisms may minimize muscle damage of myopathies due to certain RyR1 mutations.

Show MeSH
Related in: MedlinePlus