Reduced threshold for store overload-induced Ca2+ release is a common defect of RyR1 mutations associated with malignant hyperthermia and central core disease

Abstract

Mutations in the skeletal muscle ryanodine receptor (RyR1) cause malignant hyperthermia (MH) and central core disease (CCD), whereas mutations in the cardiac ryanodine receptor (RyR2) lead to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most disease-associated RyR1 and RyR2 mutations are located in the N-terminal, central, and C-terminal regions of the corresponding ryanodine receptor (RyR) isoform. An increasing body of evidence demonstrates that CPVT-associated RyR2 mutations enhance the propensity for spontaneous Ca²⁺ release during store Ca²⁺ overload, a process known as store overload-induced Ca²⁺ release (SOICR). Considering the similar locations of disease-associated RyR1 and RyR2 mutations in the RyR structure, we hypothesize that like CPVT-associated RyR2 mutations, MH/CCD-associated RyR1 mutations also enhance SOICR. To test this hypothesis, we determined the impact on SOICR of 12 MH/CCD-associated RyR1 mutations E2347-del, R2163H, G2434R, R2435L, R2435H, and R2454H located in the central region, and Y4796C, T4826I, L4838V, A4940T, G4943V, and P4973L located in the C-terminal region of the channel. We found that all these RyR1 mutations reduced the threshold for SOICR. Dantrolene, an acute treatment for MH, suppressed SOICR in HEK293 cells expressing the RyR1 mutants R164C, Y523S, R2136H, R2435H, and Y4796C. Interestingly, carvedilol, a commonly used β-blocker that suppresses RyR2-mediated SOICR, also inhibits SOICR in these RyR1 mutant HEK293 cells. Therefore, these results indicate that a reduced SOICR threshold is a common defect of MH/CCD-associated RyR1 mutations, and that carvedilol, like dantrolene, can suppress RyR1-mediated SOICR. Clinical studies of the effectiveness of carvedilol as a long-term treatment for MH/CCD or other RyR1-associated disorders may be warranted.

Keywords: Ca2+ overload; calcium release; disease mutation; malignant hyperthermia; ryanodine receptor; sarcoplasmic reticulum.

Figure 1. Co-localization of disease-associated RyR1 and RyR2 mutations in the 3D structure of RyR

The 3D structure of two RyR monomers [62,64,65] is shown. RyR1 mutations associated with MH and CCD are largely clustered in three hotspot regions: MH/CCD mutation hotspot I (highlighted in yellow), MH/CCD mutation hotspot II (in green), and MH/CCD mutation hotspot III (in cyan). MH/CCD-associated RyR1 mutations tested in the present study are highlighted in red/magenta. RyR2 mutations associated with CPVT are also clustered in these three hotspot regions. In addition to these three mutation hotspots, RyR2 also harbors another disease mutation hotspot between residues 3778–4201 (highlighted in violet) [6,9]. CPVT-associated RyR2 mutations that are co-localized with the MH/CCD RyR1 mutations tested in the present study are highlighted in black. CPVT RyR2 mutations that coincide with the MH/CCD RyR1 mutations (R164C, R2435L/H, R2454H, and P4973L) are highlighted in magenta.

Figure 2. MH/CCD-associated RyR1 mutations located in the central region of the channel reduce the threshold for SOICR

Stable, inducible HEK293 cell lines expressing RyR1 WT (A), E2347-del (B), R2163H (C), G2434R (D), R2435L (E), R2435H (F), and R2454H (G) mutants were transfected with the FRET-based ER luminal Ca²⁺-sensing protein D1ER for 48 h. The expression of RyR1 WT and mutants was induced by tetracycline 24 h before imaging. The cells were perfused with KRH buffer containing caffeine (2 mM) and increasing levels of extracellular Ca²⁺ (0–5 mM) to induce SOICR. This was followed by the addition of 1 mM tetracaine to inhibit SOICR and then 20 mM caffeine to deplete the ER Ca²⁺ store. FRET recordings from representative RyR1 WT (A) and mutant (B–G) cells (from a total of 15–91 cells) are shown. To minimize the influence by CFP/YFP cross-talk, we used relative FRET measurements for calculating the SOICR threshold (H), which was determined by the equation ((F_SOICR − F_min)/(F_max − F_min)) × 100%. F_SOICR indicates the FRET level at which SOICR occurs. The maximum FRET signal F_max is defined as the FRET level after tetracaine treatment. The minimum FRET signal F_min is defined as the FRET level after caffeine treatment. Data shown are mean ± SEM from —three to five separate experiments (*P < 0.05 vs. WT).

Figure 3. MH/CCD RyR1 mutations located in the C-terminal region reduce the SOICR threshold

Stable, inducible HEK293 cell lines expressing RyR1 Y4796C (A), T4826I (B), L4838V (C), A4940T (D), G4943V (E), and P4973L (F) mutants were transfected with the FRET-based ER luminal Ca²⁺-sensing protein D1ER for 48 h. The expression of RyR1 mutants was induced by tetracycline 24 h before imaging. The cells were perfused with KRH buffer containing caffeine (2 mM) and increasing levels of extracellular Ca²⁺ (0–5 mM) to induce SOICR. This was followed by the addition of 1 mM tetracaine to inhibit SOICR and then 20 mM caffeine to deplete the ER Ca²⁺ store. FRET recordings from representative RyR1 mutant (A–F) cells (from a total of 31–138 cells) are shown. (G) The SOICR threshold was determined by the equation ((F_SOICR − F_min)/(F_max − F_min)) × 100%. F_SOICR indicates the FRET level at which SOICR occurs. The maximum FRET signal F_max is defined as the FRET level after tetracaine treatment. The minimum FRET signal F_min is defined as the FRET level after caffeine treatment. Data shown are mean ± SEM from —three to five separate experiments (*P < 0.05 vs. WT).

Figure 4. Dantrolene suppresses SOICR in HEK293 cells expressing the central and C-terminal region MH/CCD RyR1 mutations

Stable, inducible HEK293 cells expressing the RyR1 R2136H (A), R2435H (B), or Y4796C (C) mutant were loaded with Fura-2, AM and perfused with KRH buffer containing 5 mM [Ca²⁺]_o plus 2 mM caffeine in the absence or the presence of 100 nM dantrolene. (D) The fraction of R2136H, R2435H, or Y4796C mutant cells that displayed Ca²⁺ oscillations before (control) and after the addition of 100 nM dantrolene (from a total of 39–87 cells). Data shown are mean ± SEM from three separate experiments (*P < 0.05 vs. control).

Figure 5. Carvedilol suppresses RyR1-mediated SOICR in HEK293 cells

Stable, inducible HEK293 cells expressing the RyR1 R2136H (A), R2435H (B), or Y4796C (C) mutant were loaded with Fura-2, AM and perfused with KRH buffer containing 5 mM [Ca²⁺]_o plus 2 mM caffeine in the absence or the presence of 30 μM carvedilol. (D) The fraction of R2136H, R2435H, or Y4796C mutant cells that displayed Ca²⁺ oscillations before (control) and after the addition of 30 μM carvedilol (from a total of 51–94 cells). Data shown are mean ± SEM from three separate experiments (*P < 0.05 vs. control).

Figure 6. Sustained cytosolic Ca²⁺ elevation in HEK293 cells expressing leaky MH/CCD RyR1 mutations R164C and Y522S

Stable, inducible HEK293 cells expressing RyR1 WT (A) and R164C (B), and Y523S (C) mutants were loaded with 5 μM Fura-2, AM in KRH buffer. The cells were then perfused continuously with KRH buffer containing increasing levels of extracellular Ca²⁺ (0–5 mM) plus 2 mM caffeine to induce SOICR. Fura-2 ratios were recorded using epifluorescence single-cell Ca²⁺ imaging. Traces of Fura-2 ratios of representative RyR1 WT, R164C, and Y523S cells are shown.

Figure 7. Dantrolene and carvedilol inhibit R164C- and Y523S-mediated spontaneous Ca²⁺ leak

Stable, inducible HEK293 cells expressing the RyR1 R164C (A and C) or Y523S (B and D) mutant were loaded with Fura-2, AM and perfused with KRH buffer containing 5 mM [Ca²⁺]_o plus 2 mM caffeine in the absence or the presence of 100 nM dantrolene (A and B) or 30 μM carvedilol (C and D). The fraction of R164C or Y523S mutant cells that displayed Ca²⁺ leak before (control) and after the addition of 100 nM dantrolene (E) or 30 μM carvedilol (F) (from a total of 314–417 cells). Data shown are mean ± SEM from three separate experiments (*P < 0.05 vs. control).