The RyR1 P3528S Substitution Alters Mouse Skeletal Muscle Contractile Properties and RyR1 Ion Channel Gating

Abstract

The recessive Ryanodine Receptor Type 1 (RyR1) P3527S mutation causes mild muscle weakness in patients and increased resting cytoplasmic [Ca²⁺] in transformed lymphoblastoid cells. In the present study, we explored the cellular/molecular effects of this mutation in a mouse model of the mutation (RyR1 P3528S). The results were obtained from 73 wild type (WT/WT), 82 heterozygous (WT/MUT) and 66 homozygous (MUT/MUT) mice with different numbers of observations in individual data sets depending on the experimental protocol. The results showed that WT/MUT and MUT/MUT mouse strength was less than that of WT/WT mice, but there was no difference between genotypes in appearance, weight, mobility or longevity. The force frequency response of extensor digitorum longus (EDL) and soleus (SOL) muscles from WT/MUT and MUT/MUT mice was shifter to higher frequencies. The specific force of EDL muscles was reduced and Ca²⁺ activation of skinned fibres shifted to a lower [Ca²⁺], with an increase in type I fibres in EDL muscles and in mixed type I/II fibres in SOL muscles. The relative activity of RyR1 channels exposed to 1 µM cytoplasmic Ca²⁺ was greater in WT/MUT and MUT/MUT mice than in WT/WT mice. We suggest the altered RyR1 activity due to the P2328S substitution could increase resting [Ca²⁺] in muscle fibres, leading to changes in fibre type and contractile properties.

Keywords: Ca2+ activation of skinned muscle fibres; RyR1 P3528S substitution; fibre-type composition; mouse model; muscle contractile properties; ryanodine receptor; single ryanodine receptor activity.

Figure 1

Effects of the RYR1 P3528S mutation on the appearance, ambulation rate and strength of WT/MUT and MUT/MUT mice compared to their WT/WT littermates. (A) Representative photographs of young mice taken immediately after CO₂ euthanasia. From left to right: a WT/WT male, a WT/MUT female and a MUT/MUT male. (B,C) Average ambulation rate (m/min, left column), length of time that mice held onto the grid bar before letting go (hold duration (min) centre column) and the ability of mice to lift chains of increasing weight for 3 s or less, calculated as a weight score (detailed in the Section 4) (in arbitrary units (AU), right column). Data are separated into age groups with mice aged between 1.5 and 6 months in (A) and between 7 and 20 months in (B). The data were obtained from the same mice as those listed in Table 1. The number of mice in each group are given in Table 1. The data are shown as mean ± sem. The black asterisks indicate significant differences from between males and females. The red asterisks indicate a significant difference from WT/WT.

Figure 2

Effects of the RYR1 P3528S mutation on the contractile properties of EDL and SOL muscles from WT/MUT and MUT/MUT mice compared to their WT/WT littermates. The force response of EDL muscles (A) and SOL muscles (B), expressed as a % of Fmax in response to increasing stimulation frequency. Force output produced by EDL muscles (C) and SOL muscles (D), normalized to muscle cross-sectional area (specific force, sFmax). * p < 0.05—MUT/MUT mice compared their WT/WT littermates, as determined by two-way ANOVA; # p < 0.05—MUT/MUT and WT/MUT mice compared WT/WT mice, as determined by one-way ANOVA. WT/WT mice (N = 6), WT/MUT mice (N = 4) and MUT/MUT mice (N = 5).

Figure 3

Typical example of a type II (Ai) and type I fibre (Aii) obtained from the same SOL muscle of a wild type mouse (WT/WT). Force responses elicited when directly activating the contractile apparatus with heavily Ca²⁺-buffered solutions with progressively higher free [Ca²⁺] (grey ticks under the black force trace indicate the pCa of successive solutions: >9.00, 6.70, 6.40, 6.22, 6.02, 5.88, 5.75, 5.48, 4.50 then back into >9.00 (repeated to test reproducibility)). A maximum Ca²⁺-activated force “max” response was elicited first before exposing the fibre segment to pSr 5.3. (Bi) “Mixed” fibre force recording of a SOL muscle obtained from a WT/MUT mouse. Note “force oscillations” at ~40% force in the first staircase and a plateauing of force around ~60% maximum Ca²⁺-activated force. Stimulus artefacts caused when transferring the fibre segment from one bath to the next, are truncated. (Aiii) Hill “h” fits to the force pCa staircases from Ai (pCa₅₀ = 5.883 pCa units and h = 4.429) and (Aii) (pCa₅₀ = 6.023 pCa units and h = 2.716). Similarly, (Bii) shows h fits for three consecutive fibres, one obtained from an EDL muscle and two from a SOL muscle from one WT/MUT mouse. The responses of these fibres verify that the changes in Ca²⁺ sensitivity are a manifestation of the physical properties of the fibre’s myosin content, not errors associated with the test solutions or other possible experimental variables. The pCa₅₀ and h values in Bii are 5.901 pCa units and 2.625 (SOL type 1 fibre; red curve), 5.858 pCa units and 4.702 (EDL type II fibre; blue curve), and 5.816 pCa units and 2.958 (SOL mixed fibre, black curve), respectively. Note: Hill fits in (Aiii,Bii) are for the individual fibres not the average values.

Figure 4

Typical examples of a type II fibre obtained from an EDL muscle in (Ai,Aii) and a type I fibre from a SOL muscle in (Bi,Bii) from one MUT/MUT mouse, with generalized responses shown in (Aiii,Biii). In both (Ai,Bi) panels, the maximum “max” force responses were elicited after exposing the fibre segment to pCa 4.5 and then the pSr 5 response was evoked. This was followed by two force “staircases” elicited by directly activating the contractile apparatus with heavily Ca²⁺-buffered solutions with progressively higher free [Ca²⁺] (grey ticks under the black force trace indicate the pCa of successive solutions: >9.00, 6.70, 6.40, 6.22, 6.02, 5.88, 5.75, 5.48, 4.50 then back into >9.00). Panels (Aii) and (Bii) are Hill “h” fits to the force pCa staircases from (Ai) and (Bi), respectively. The force traces in (Ai,Bi) in are from two fibres sequentially tested on the same day using the same solutions. The responses verify that the changes in Ca²⁺ sensitivity were a manifestation of the physical properties of the fibre’s myosin content, not errors associated with the test solutions or other possible experimental variables. Generalized shifts in Ca²⁺-sensitivity in EDL and SOL fibres from MUT/MUT mice are shown in panels (Aiii,Biii).

Figure 5

Single RyR1 channel activity in WT/WT channels increases with age and the effect of reducing Ca²⁺ from 1 µM to 300 nM is significant only in MUT/MUT channels. (A–C) Representative 10 s records from WT/WT RyR1 channels showing the age-dependent changes in activity recorded with cytoplasmic (cis) [Ca²⁺] of 1 µM (left), then 300 nM (middle) and then after increasing cytoplasmic [caffeine] to 10 mM while the cis [Ca²⁺] was maintained at 300 nM (right). Recordings are from young (A), middle-aged (B) and old (C) WT/WT mice. Channel activity is shown at +40 mV, with openings upward from the closed state at the bottom of each single channel record. The records in each row were obtained from the same RyR1 channel. P_o values are given above each record. (D–F) Graphs of average channel open probability (P_o) with 1 µM cis Ca²⁺ (C1), 300 nM cis Ca²⁺ (C2) and then following progressive increases in cytoplasmic [caffeine] to 10 µM, 100 µM, 1 mM and 10 mM. The inserts in each graph are expanded to show the effects of reducing cis [Ca²⁺] from 1 µM (C1) to 300 nM (C2). Average P_o is shown for channels from WT/WT (left), WT/MUT (middle) and MUT/MUT (right) mice from the young (D), middle-aged (E) and old (F) groups. Data are shown as mean ± sem. The average values include P_o at +40 mV and −40 mV. The number of observations (n) for each group are as follows: young WT/WT, n = 14; young WT/MUT n = 16; young MUT/MUT n = 12; middle-aged WT/WT n = 16; middle-aged WT/MUT n = 16; middle-aged MUT/MUT n = 14; old WT/WT n = 8; old WT/MUT n = 8; old MUT/MUT n = 6. * or *, significantly different from C2 (300 nM cis Ca²⁺); ^@, significantly different from C1 (1 µM cis Ca²⁺) in channels from young WT/WT mice; ^@, significantly different from C2 (300 nM cis Ca²⁺) in channels from young WT/WT mice.

Figure 6

The activity of individual MUT/MUT RyR1 channels is higher with 1 µM cis Ca²⁺ than with 300 nM cis Ca²⁺ in most situations and the average relative P_o is significantly greater in channels from old MUT/MUT mice than in channels from old WT/WT mice. (A,B) Representative 10 s records from MUT/MUT RyR1 channels from each age group showing reduced activity with a reduction in cytoplasmic [Ca²⁺] from 1 µM (A) to 300 nM (B). The channels in (A,B) were isolated from young (left), middle-aged (centre) and old (right) MUT/MUT mice. Channel activity is shown at +40 mV, with openings upward from the closed state at the bottom of each single channel record. The recordings in (A,B) in each age group are from the same channel. The P_o for each record is shown above the record and the corresponding relative P_o values are given in (A). (C–E) Graphs of average open probability relative to C2 (300 nM cis Ca²⁺) (rel P_o) are plotted for 1 µM cis Ca²⁺ (C1), and after progressive increases in cytoplasmic [caffeine] to 10 µM, 100 µM, 1 mM and 10 mM. The inserts in each graph are expanded to show the effects of reducing cis [Ca²⁺] from 1 µM (C1) to 300 nM (C2). Average rel P_o is shown for channels from WT/WT (left), WT/MUT (middle) and MUT/MUT (right) mice, in young (C), middle-aged (D) and old (E) groups. The average values include rel P_o at +40 mV and −40 mV. The number of observations (n) for each group are as follows: young WT/WT, n = 14; young WT/MUT n = 16; young MUT/MUT n = 12; middle-aged WT/WT n = 16; middle-aged WT/MUT n = 16; middle-aged MUT/MUT n = 14; old WT/WT n = 8; old WT/MUT n = 8; old MUT/MUT n = 6. * or *, significantly different from C2 (300 nM cis Ca²⁺); ^#, significantly different from rel P_o of old WT/WT channels.

Figure 7

The cross-sectional profile of fibres differs in EDL muscles from WT/MUT and MUT/MUT mice compared with that of WT/WT mice. (A,B) show representative micrographs of EDL (A) and SOL (B) muscles from WT/WT mice (left), WT/MUT mice (centre) and MUT/MUT mice (right) aged between 2 and 4 months. The EDL muscles contained two distinct populations of fibres: larger EDL group A fibres and smaller EDL group B fibres. Smaller EDL group B fibres in (A) are marked with a yellow dot. The magnification is the same for panels (A,B). The scale bar below the right image in panel (A) applies to all images in (A,B). (C,D) show graphs of the average % of fibres in each group in EDL and SOL muscles from mice from each of the three genotypes aged 2–4 months (C) and 6–12 months (D). The cross-sectional areas of ~34–80 fibres were measured in each muscle and the fibres were allocated to group A or group B. The average % fibres in each group is shown as mean ± sem. *, significant difference between the % of fibres from WT/WT mice and WT/MUT or MUT/MUT mice. ^@, significant difference between the % of fibres from WT/MUT and MUT/MUT mice. Number of mice in the 2–4-month-old group (C): WT/WT, 17; WT/MUT, 14; MUT/MUT, 11. Number of mice for the 6–12-month-old group (D): WT/WT, 3; WT/MUT, 5; MUT/MUT, 11.

Figure 8

Silver-stained SDS-PAGE gels confirm fibre types assigned to individual fibres in histological sections of EDL and SOL muscles. (A–D) show bands between 150 kDa and 250 kDa in representative silver-stained SDS-PAGE gels of muscle homogenates. The image in (A) is an expansion of lanes 3, 4 and 5 lanes of the gel shown in (B) in order to compare bands in mouse homogenates (in this case MUT/MUT, lane 3) with those in rat EDL (lane 4) and rat SOL (lane 5) samples. The expansion illustrates the assignment of myosin isoforms to the bands in mouse homogenates, irrespective of genotype. The higher molecular mass IIA and IIX bands are well defined in rat EDL, while the IIA band is absent from rat SOL and from mouse limb muscles. (B–D) Comparison of myosin isoform in mice of the 3 genotypes and show the lack of a genotype dependence. (B). Hindlimb muscles from older (18–19 months) WT/WT, WT/MUT and MUT/MUT mice (lanes 1–3) and rat (~6 months) EDL and SOL muscles (lanes 4 and 5). (C). Hindlimb muscles of younger (7–8 months) WT/WT, WT/MUT and MUT/MUT mice (lanes 1 to 3) and 6-month-old rat EDL and SOL muscles (lanes 4 and 5). (D). Forelimb muscles from young (2 months, lanes 4–6) and old (19–20 months, lanes 1–3) WT/WT, WT/MUT and MUT/MUT mice. (A–D) Mouse muscle homogenates were obtained from 2 mice of the same age and genotype. The mouse homogenate pellets were resuspended at a concentration of 0.6 mg/mL. Rat muscle homogenates are from one rat. The rat homogenate pellets were resuspended at a concentration of 0.3 mg/mL. A 20 µL volume of the resuspended homogenate pellet solution was loaded onto the gels in all cases (A–D). The position of the 150 kDa and 250 kDa molecular weight markers are shown on the left-hand side of each gel.