Skeletal muscle physiology of mice with RyR1 mutation; Preparing for drug discovery

Calcium homeostasis in muscle is crucial for function. Ryanodine receptors, such as RyR1, regulate calcium release into skeletal muscle fibers. Alterations in ryanodine receptors, genetic or post-translational, impair muscle force generation and contractility. Sensitive physiological measures are critical for testing treatments of muscle disorders with dysregulated calcium. Our objective is to identify sensitive contractility measures in muscle of mice with genetically altered RyR1 proteins. We hypothesized that mice with one mutated RYR1 allele would have intermediate skeletal muscle force-generating and contractile properties compared to those with zero or two mutated RYR1 alleles, and that properties dependent upon calcium dynamics would be most affected. We used a knock-in mouse model carrying the malignant hyperthermia–associated RyR1 p.G2435R missense mutation in MH region 2. These mice display increasingly severe RyR1 calcium leak in skeletal muscle fibers in a gene-dosage dependent manner. In vitro physiological measurements of baseline force production and contractile dynamics were performed on live whole soleus muscle from homozygous (RYR1WT/WT; HOM), heterozygous (RYR1KI/WT; HET), and wild-type littermates (RYR1WT/WT; WT). Mice ranged in age from 14-18 wk, and each group consisted of n=5-9. All contractility metrics were performed at 25°C. Key outcomes include maximal and submaximal force generation as well as contraction and relaxation dynamics. All data were analyzed using one-way ANOVA with Holm-Sidak post hoc analysis. For maximal tetanic force generation, female HOM mice generated 78% less isometric, 42% less concentric, and 61% less eccentric forces than WT (p≤0.033). Female HET generated 31% more isometric, 54% more concentric, and 30% more eccentric forces than WT (p≤0.008). HOM mice reached 50% of their maximum force at 27.5 Hz, while WT and HET mice reached 50% of maximum force at ~10 Hz (p≤0.001). Rates of isometric tetanic contraction and relaxation were also different between genotypes. Compared to WT, the rate of contraction was 36% faster for HET and 80% slower for HOM mice (p≤0.001). The rate of relaxation was 28% faster for HET and 79% slower for HOM mice (p≤0.006). When normalized to peak twitch force, HOM mice had 400% greater time-to-peak twitch and 150% greater half relaxation time than WT (p≤0.001). In male mice, no significant differences were measured between WT and HET mice in baseline force production or contractile dynamics (p≥0.387). Similar to females, HOM males had up to 83% lower tetanic and twitch forces (p≤0.001), and 88% slower rates of contraction and relaxation (p≤ 0.001). Male HOM mice reached 50% of their maximum force at 33.3 Hz, while WT and HETs reached 50% of maximum force at ~18.5 and 16.8 Hz, respectively (p≤0.001). This data reveals how altered RyR1 calcium leak impacts force generation and muscle contractility in a genotype-specific manner. This research will provide a sensitive, physiologically relevant platform for identifying therapeutic drugs that target muscle disorders by correcting calcium leak. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.