A Novel Recessive Mutation in Cardiac Ryanodine Receptor 2 Causes Loss of Function, Leading to Ventricular Fibrillation and Sudden Death

Background: The cardiac ryanodine receptors (RyR2) are calcium (Ca²⁺) release channels present in the sarcoplasmic reticulum (SR) which play a major role in regulation of Ca²⁺ homeostasis in the heart. We recently discovered a novel RyR2 missense mutation, G3118R, with recessive co-segregation, in a family presenting with cardiac arrest and ventricular fibrillation. The mechanism by which this mutation determines this unusual clinical phenotype is unknown.

Aims: The aims of this study were to perform functional analysis of G3118R RyR2 in order to investigate the mechanism of this mutation`s pathogenicity.

Methods: Caffeine induced calcium release assays (1–100 mM, normalized to the peak amplitude for maximal Ca²⁺ release induced in each experiment) were performed in HEK-293 cells expressing G3118R missense mutation and in wild type (WT) RyR2. Store-Overload-Induced Calcium Release (SOICR) was measured using single-cell Ca²⁺ imaging with the FRET-based endoplasmatic reticulum (ER) luminal Ca²⁺-sensing protein D1ER.

Results: G3118R HEK-293 cells reached peak fluorescence amplitudes at a higher caffeine concentration compared with WT RyR2 (40mM versus 20mMwhich indicated a decrease in RyR2 channel Ca²⁺ ‏ release. In addition, adding Ca²⁺ to the extracellular environment induced spontaneous ER Ca²⁺ oscillations in both wild type and mutant RyR2, which is known as SOICR. However, in G3118R mutant cells the fractional Ca²⁺ release during SOICR (activation threshold−termination threshold) was significantly lower compared to RyR2 WT (35% n=67 versus 47% n=16, p<0.01), whereas the ER Ca²⁺ store capacity (F_max−F_min) was significantly higher (74% n=67 versus 54% n=16, p<0.01). These results are compatible with reduction of Ca²⁺ release in response to increase in luminal Ca²⁺.

Conclusion: We report a unique loss of function mechanism of RyR2 channel dysfunction, caused by a novel recessively inherited mutation, associated with ventricular fibrillation and sudden death.