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

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.

Keywords: KATP channel; cell biology; congenital myopathy; human; mouse; potassium homeostasis; ryanodine receptor.

Figure 1.. ENU-induced Ryr1^AG mutation mimics clinical and pathological features of CCD in heterozygous mice.

(A) Missense mutation in exon 93 of Ryr1 changes A to G, resulting in substitution of glutamic acid with glycine (E4242G). (B) Average grip strength assayed using vertical digital push–pull strain gauge on Ryr1^+/+ and Ryr1^AG/+ mice at different ages raised on control 0.6% potassium diet. (C) In vivo hanging task determination of upper-body strength of Ryr1^+/+ and Ryr1^AG/+ mice at different ages (B, C, 10 trials/mouse, n = 5 per set). (D, E) H&E staining indicate central nuclei (arrows) in Ryr1^AG/+ (E) but not Ryr1^+/+ (D) vastus lateralis muscle from 1-year old mice raised on control 0.6% potassium diet. (F, G) NADH-TR staining indicates cores (white arrows) in 1-year old vastus lateralis of RyR1^AG/+ but not Ryr1^+/+. (H, I) Cytochrome oxidase (COX) staining denotes a decrease in mitochondrial function (white arrows) in vastus lateralis muscle of Ryr1^AG/+ (I) compared to Ryr1^+/+ (H). Scale bar = 50 μm. (J–L) Transmission electron microscopy of 2-month old Ryr1^AG/+ soleus muscle. (J, K) Regions of Z line streaming and associated sarcoplasmic disruption and cores (white arrows) of myofibrils. (L) Enlarged T-tubules are present in type I fibers (scale bars: J = 1 μm; K = 2 μm; L = 0.2 μm ). DOI: http://dx.doi.org/10.7554/eLife.02923.003

Figure 2.. Calcium homeostasis is disrupted in Ryr1^AG/+ muscle.

Fura-2 ratiometric imaging of myoplasmic Ca²⁺ in 2-month old muscle. (A) Ratiometric analysis of 4-CmC sensitivity in soleus muscles of wild-type (black) and Ryr1^AG/+ littermates (grey). (B) Average ratiometric analysis of myoplasmic Fura-2 signal normalized to wild-type signal (n above bar represents number of analyzed fibers). (C) Representative fura-2 ratiometric signal of muscle fibers showing percent change after addition of 20 μM thapsigargin (TG) from wild-type (black) and Ryr1^AG/+ (grey) littermate. (D) TG induced maximum response in muscle fibers (n above bar represents number of analyzed fibers). (E, F) Representation and quantification of Mn²⁺ quenching of Fura-2 fluorescence ratiometric signal illustrating increased influx of store operated calcium entry in soleus muscle fibers of Ryr1^AG/+ (grey) muscle fibers compared to wild-type (black) muscle fibers (dash lines represent slope). In (E), arrows indicate when media was introduced with 0 Ca²⁺ followed by 0.5 mM Mn²⁺. DOI: http://dx.doi.org/10.7554/eLife.02923.006

Figure 3.. Defects in mitochondrial function in Ryr1^AG/+ mice.

(A–C) Representative isolated fiber with Rhod2 fluorescence of 2-month old Ryr1^AG/+ in 3 mM KCl (A) and 1 min (B) and 10 min (C) after application of 1 mM 4-CmC (punctate fluorescence are calcium marks). (D) Quantification of mitochondrial calcium marks visualized with Rhod2-AM from isolated soleus muscle fibers from Ryr1^+/+ (black bars) and Ryr1^AG/+ (gray bars) mice. (E) Superoxide labeling of isolated muscle fibers from Ryr1^+/+ and Ryr1^AG/+ mice. (F) Intensity of TMRE labeling of isolated soleus muscle fibers from Ryr1^+/+ and Ryr1^AG/+ littermates with saponin. The numbers on top of bars in graphs represent number of fibers examined. (G) ATP content from Ryr1^+/+ and Ryr1^AG/+ isolated soleus muscle from age-matched mice (n = 4 for each age and group). Scale bar = 5 μm. DOI: http://dx.doi.org/10.7554/eLife.02923.007

Figure 4.. Detection and compensation of an internal potassium leak in RyR1^AG/+ muscle.

Fluorescence imaging of PBFI at 340 nm in Ryr1^+/+ (A) and Ryr1^AG/+ (B) soleus muscle in 3 mM Ringer's solutions. (C) Ratiometric potassium imaging obtained at 340 and 380 nm wavelengths provided a ratio of fluorescence in Ryr1^+/+ (black) and Ryr1^AG/+ (grey) soleus muscle (normalized to Ryr1^+/+). (D) Representation of the ratiometric imaging experimental paradigms used in Figure 4 showing bath applications of 3 mM KCl, 7 mM KCl, 0 mM KCl, and 3 mM KCl with 2 μM glibenclamide in Ringer's solutions. (E) Slope of intracellular K⁺ fluorescence intensities in experimental conditions. (F, H) Normalized intracellular K⁺ concentration in Ryr1^+/+ (black) and Ryr1^AG/+ (grey) soleus muscle in 3 mM KCL (F, H) compared to soleus from contralateral limb in 7 mM KCl Ringer's solutions (F) or 3 mM KCL plus 2 μM Glibenclamide (H) (muscle was bathed in solutions for 1.5 hr before imaging, n is the number of fibers examined from four mice). (G) Rate of change in PBFI fluorescence after acute bath application of 2 μM Glibenclamide. Scale bar = 20 μm. DOI: http://dx.doi.org/10.7554/eLife.02923.008

Figure 4—figure supplement 1.. Serum level measurements from Ryr1^+/+ (black bars) and Ryr1^AG/+ (grey bars) in 2- and 6-month old mice.

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

Figure 5.. Altered activity of K_ATP channels involved in potassium transport.

(A) Western blots of membrane enriched lysate (that lacks mitochondria, peroxisomes, and lysosomes) from 2-month old wild-type and heterozygous Ryr1^AG/+ soleus muscle analyzed for K_ATP6.1, K_ATP6.2, and K_IR2.1. (B) Quantification of protein levels from Western blots of membrane enriched lysates. Each sample was first normalized to its own loading control and then the values from mutant and wild-type on the same blot were compared. Statistical analyses were determined from a minimum of 3 blots and at least two independent samples (n = 4 for each Western). (C) K_ATP current densities of isolated soleus muscle fibers from 2-month old wild-type and Ryr1^AG/+ littermates with and without glibenclamide. Numbers on top of bars are number of fiber recordings. (D–E) Representative current density recordings with the same tip resistance from Ryr1^+/+ and Ryr1^AG/+ soleus muscle fibers (E) with and (D) without 2 μM glibenclamide. DOI: http://dx.doi.org/10.7554/eLife.02923.010

Figure 6.. Increased potassium diet can rescue muscle strength and reverse the CCD histology and myopathy.

(A) Normalized internal potassium concentrations of soleus muscle from 2-month old Ryr1^+/+ and Ryr1^AG/+ mice fed 0.6% K⁺ diet for 4 weeks, then placed on 5.2% K+ diet or 0.6% diet + enalapril for 4 weeks, and then bath exposed to different extracellular potassium concentrations. (B, C) Average grip strength (B, five trials/mouse and 5 mice/set; p values <0.001) and in vivo hanging task (C, 10 trials/mouse, n = 5 per set; p values <0.001) assayed from 2-month old Ryr1^+/+ (black bar) and Ryr1^AG/+ (grey bar) mice maintained for 4 weeks on control 0.6% K⁺ diet, 5.2% K⁺ diet, or 0.6% K⁺ diet supplemented with enalapril. (D) Quantification of number of internalized nuclei per 100 myofibers in 12-month old mice or in 2-month old mice maintained for 4 weeks on control 0.6% K⁺ diet, 5.2% K⁺ diet, or 0.6% K⁺ diet supplemented with enalapril (n = 10 per muscle, n = 3 per set of muscles for total of n = 30; p values <0.001). (E–P) Vastus lateralis myofibers from 2-month old Ryr1^+/+ and Ryr1^AG/+ mice maintained for 4 weeks on control 0.6% K⁺ diet, 5.2% K⁺ diet, or 0.6% K⁺ diet supplemented with enalapril. Cross-sections stained with H&E (left panels) and COX (right panels). Ryr1^AG/+ mice on 5.2% K diet show increased COX staining and no internalized nuclei, similar to Ryr1^+/+. These pathological features are still observed in Ryr1^AG/+ mice on 0.6% K⁺ diets. Enalapril increases COX staining but some internalized nuclei are observed, even in Ryr1^+/+. Scale bar = 50 μm; error bars as standard error of the mean (SEM). DOI: http://dx.doi.org/10.7554/eLife.02923.012

Figure 6—figure supplement 1.. Blood pressure measurements from Ryr1^+/+ (black bars) and Ryr1^AG/+ (grey bars) in 2-month old mice.

Non-invasive measurements of systolic (A) and diastolic (B) blood pressure were performed using a Visitec-2000 tail-cuff apparatus. Measurements were taken for 3 days to acclimatize the animals for a more reproducible blood pressure measurement and then the data were collected on day 4. DOI: http://dx.doi.org/10.7554/eLife.02923.013

Figure 7.. Increased potassium diet influences K_ATP channel activity and mitochondrial function.

(A) Membrane enriched lysate (lacking mitochondria, peroxisomes, and lysosomes) from soleus muscle of 2-month old Ryr1^+/+ and Ryr1^AG/+ mice fed 5.2% potassium supplemented diet for 4 weeks analyzed by Western blot for K_ATP6.1, K_ATP6.2, and K_IR2.1 (* is p < 0.001). (B) K_ATP current densities of isolated soleus muscle fibers from Ryr1^+/+ and Ryr1^AG/+ littermates after 4 weeks on 5.2% K diet compared to 0.6% K diet. (C) ATP content from Ryr1^+/+ and Ryr1^AG/+ isolated soleus muscle from mice on 0.6% or 5.2% potassium diet. (D) Quantification of calcium marks visualized with Rhod2-AM from isolated soleus muscle from Ryr1^+/+ and Ryr1^AG/+ mice with and without diet therapy. (E) Superoxide labeling of isolated soleus muscle from Ryr1^+/+ and Ryr1^AG/+ mice with and without therapy. Error bars as standard error of the mean (SEM). DOI: http://dx.doi.org/10.7554/eLife.02923.014

Figure 8.. Inhibition of K_ATP channels can reverse the histological and myopathic phenotypes.

(A, B) Average grip strength (A, five trials/mouse and 5 mice/set; p values <0.001) and in vivo hanging task (B, 10 trials/mouse, n = 5 per set; p values <0.001) assayed from 2-month old Ryr1^+/+ (black bar) and Ryr1^AG/+ (grey bar) mice maintained for 4 weeks on 0.6% K diet and glibenclamide (15 mg/kg/day). (C) ATP content from soleus muscle of Ryr1^+/+ and Ryr1^AG/+ mice after 4 weeks on 0.6% K diet without or with glibenclamide (n = 4 muscles per condition). (D) Quantification of calcium marks visualized with Rhod2-AM from isolated soleus muscle from Ryr1^+/+ and Ryr1^AG/+ mice with and without glibenclamide therapy. (E) Normalized internal potassium concentrations after glibenclamide therapy. (F) K_ATP current densities of isolated soleus muscle fibers from Ryr1^+/+ and Ryr1^AG/+ littermates after 4 weeks on glibenclamide therapy compared to 0.6% K diet. (G) Quantification of number of internalized nuclei per 100 myofibers in all conditions (n = 10 per muscle, n = 3 per set of muscles for total of n = 30; p values <0.001). (H–K) Vastus lateralis myofiber from Ryr1^+/+ and Ryr1^AG/+ mice maintained for 4 weeks on glibenclamide. Arrow in Ryr1^+/+ myofiber (F) shows the rare occurrence of internal nuclei. Cross-sections stained with H&E (left panels) and COX (right panels). Ryr1^AG/+ mice on glibenclamide show increased COX staining, similar to Ryr1^+/+. Scale bar = 50 μm; error bars as standard error of the mean (SEM). DOI: http://dx.doi.org/10.7554/eLife.02923.015

Figure 9.. Relative variation of quantitative qRT-PCR of human control RNA and RNA from human muscle biopsies.

Whisker plots derived from the data in Table 2 of control muscle (yellow), congenital myopathy (CCD) with a mutation in RyR1 (green), and without a mutation in RyR1 (white). (A) ABCC8, (B) ATPA1, (C) KCNJ2, (D) KCNJ8, (E) KCNJ11, (F) PRKAA1. Values normalized to GAPDH before normalization to pooled control human muscle RNA. DOI: http://dx.doi.org/10.7554/eLife.02923.017