Statins are cornerstone therapies to decrease cholesterol and prevent cardiovascular disease. Their clinical utility is limited by statin-associated muscle symptoms (SAMS), whose molecular origins have remained poorly defined. Statins act by inhibiting the HMG-CoA reductase. This mechanism does not fully explain the muscle toxicity, specifically because inactive statin forms and decreased drug concentrations can provoke symptoms. The aim of this study was to evaluate whether skeletal muscle ryanodine receptor (RyR1), a central regulator of Ca²⁺ release and muscle contraction, serves as a direct off-target of statins and contributes to SAMS, and to assess how genetic susceptibility modifies this effect.
The researchers used an integrated experimental method in combination with structural biology, biochemical, and electrophysiological assays, in vivo mouse models, and rigorous statistical analysis. Cryo–electron microscopy (cryo-EM) was used to evaluate high-resolution structures of endogenous RyR1 purified from mouse skeletal muscle in the presence and absence of simvastatin. Radioligand binding assays with [³H]simvastatin and [³H]ryanodine quantified binding affinities and channel activation
Planar lipid bilayer recordings measured RyR1 and RyR2 single-channel activity in response to increasing the simvastatin concentrations. Physiological relevance was tested in wild-type (WT) mice and heterozygous RyR1-T4709M (TM) knockin mice treated with simvastatin with or without the RyR-stabilizing Rycal drug S107. Statistical analyses were performed by using two-tailed Student’s t tests or two-way ANOVA with proper post hoc comparisons. Data were expressed as mean ± SEM, and P < 0.05 was considered statistically significant.
Cryo-EM analysis revealed two distinct simvastatin binding sites per RyR1 protomer in the transmembrane pore area: a high-affinity site (Sim-1) between S3 and S4 helices and a lower-affinity site (Sim-2) near the luminal end of the S6 helix. Radioligand binding assays showed a biphasic dosage response curve consistent with two binding sites, which yield a dissociation constant of about 0.7 μM for the high-affinity site and ~45 to 50 μM for the low-affinity site (R² ≈ 0.99). Mutations disrupting the Sim-1 significantly elevated EC₅₀ and decreased maximal activation in [³H]ryanodine binding assays. Mutation at Sim-2 mainly decreased maximal effect (Emax), which confirms the distinct functional contributions of each site.
Simvastatin elevated RyR1 open probability in a dose-dependent manner from 0.2% at baseline to 2 to 4% at 1 to 10 μM and 46% at 100 μM simvastatin in single channel recordings. It was consistent with statistically significant channel activation. This same activation profile was seen for RYR2 with no detectable inhibition under the tested conditions.
Statistical comparison of muscle function showed that simvastatin-treated WT mice exhibited significantly increased maximal specific force in extensor digitorum longus and soleus muscles (about 22 to 25% elevation at 120 Hz, P < 0.05 to P < 0.001) as compared to placebo, with no major differences in grip strength, fatigue, or serum creatine kinase levels. Heterozygous TM mice showed a major reduction in diaphragm-specific force after simvastatin treatment (29% decrease at peak stimulation, P < 0.001) and changes in limb muscles trended downward but did not reach statistical significance. Two-way ANOVA showed that cotreatment with S107 completely prevented simvastatin-induced diaphragm weakness in TM mice, restoring force production to placebo levels without changing muscle performance in WT mice.
This study gives statistically robust structural, functional, and physiological evidence that simvastatin directly binds and activates RyR1 by two conserved transmembrane sites. It established RyR1 as a key mediator of statin-associated muscle effects. WT muscle tolerates this activation and may even exhibit enhanced force. Genetically sensitized RyR1 channels, like those carrying the TM mutation, show statistically significant susceptibility to statin-induced dysfunction. These results suggest that SAMS can increase from RyR1 dysregulation in the predisposed individuals and highlight the RyR1 stabilization or rational statin redesign as a promising method to decrease muscle toxicity without compromising lipid-lowering efficacy.
Reference: Weninger G, Dridi H, Reiken S, et al. Structural basis for simvastatin-induced skeletal muscle weakness associated with type 1 ryanodine receptor T4709M mutation. J Clin Invest. 2025;135(24):e194490. doi:10.1172/JCI194490
