DASARI, SURENDRA

Abstract Background Calcium release channel deficiency syndrome (CRCDS) arises from loss-of-function variants in the RYR2-encoded type 2 ryanodine receptor (RyR2), predisposing patients to sudden cardiac arrest/death (SCA/SCD) despite normal exercise testing. Previously, a novel homozygous RYR2 duplication causing ~ 80% RyR2 reduction was associated with exertion-related SCD. However, how Ca2+ homeostasis is maintained despite RyR2-loss remains unknown, as RYR2-/- animals are non-viable. Purpose To generate a RYR2-knockout (RYR2-KO-/-) induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model to dissect compensatory mechanisms sustaining Ca2+ homeostasis and identify arrhythmogenic pathways predisposing to SCD. Methods CRISPR/Cas9 editing was used to introduce a homozygous c.163delT variant into a wild-type iPSC line (control), generating a RYR2-KO-/- model. A patient-derived RYR2-duplication (RYR2-DUP+/+) iPSC line was used for comparison. RyR2 expression was assessed by western blot (WB) and immunofluorescence (IF). Proteome and phospho-proteome were assessed using omics approaches. Intracellular Ca2+ handling was evaluated by Fluo-4 AM imaging (0.5 Hz and long-burst–long-pause [LBLP] protocol). Electrophysiological properties (action potential [AP], and sarcolemmal currents [ICaL, INa and INCX]) were assessed by patch-clamp. Results Despite complete RyR2-loss in RYR2-KO-/- iPSC-CMs as confirmed by WB and IF, contractility and contractile machinery expression was preserved. Ca2+ transient amplitude (ΔF/F0) was reduced markedly in RYR2-KO (0.14±0.01, p<0.01) and RYR2-DUP (0.27±0.2, p<0.01) compared to control (0.52±0.02). Proteomics revealed a significant NCX1 up-regulation and IP3R-interacting protein (ITPRIP) down-regulation in RYR2-KO compared to control, with pathway-analyses showing significant up-regulation of IP3-related pathways. Pharmacological IP3Rs block (2-APB, 10μM) abolished Ca2+ transient generation in RYR2-KO iPSC-CMs (Amplitude: baseline: 0.15±0.01, 2-APB: 0.06±0.01, p<0.01). RyR2-ablation induced electrical remodeling (pA/pF), including ICaL reduction, INCX up-regulation and hyperpolarization of the INa window current in RYR2-KO (ICaL [at 0mV]: -8.5±1.6; INCX [at -110mV]: -4.1±1.1) as compared to control (ICaL:-7.5±3.7, p<0.01; INCX:-1.5±0.5, p=0.02). RYR2-KO CMs demonstrated increased delayed after-depolarizations and beat-to-beat variability as compared to control. Despite Ca2+ sparks abolishment, RYR2-KO CMs displayed higher erratic beating (at 0.5Hz) and arrhythmogenic propensity than control, showing ventricular tachycardia-like events during LBLP stimulation. Conclusions Genetic RYR2-ablation disrupts physiological Ca2+ cycling and triggers electrical remodeling, including INCX up-regulation, overall promoting arrhythmogenesis. Compensatory IP3Rs activation sustains Ca2+ transient generation, revealing a mechanism preserving excitation-contraction coupling that may paradoxically promote arrhythmogenesis in CRCDS.RYR2-KO: Generation and CharacterizationElectrophysiological Assessment

Objective To estimate the prevalence, incidence, and disease burden of autoimmune seizures and epilepsies in Olmsted County. Methods We conducted a retrospective, population‐based study using the Rochester Epidemiology Project (1996–2019). Age‐ and sex‐standardized rates (2019 United States standard population) per 100,000 were calculated with 95% confidence intervals (95% CIs). Temporal trends in incidence were assessed over 3 consecutive 8‐year intervals (1996–2003, 2004–2011, and 2012–2019) using Poisson regression to estimate rate ratios per period and trend. Disability‐adjusted life years (DALYs), years of life lost (YLL), and years lived with disability (YLD) and 95% uncertainty intervals (95% UIs) were estimated for 2019. Results Twenty‐three patients were identified between 1996 and 2019. In 2019, the standardized prevalence of all autoimmune seizures and epilepsies was 9.81 (95% CI, 5.59–15.21) per 100,000, including autoimmune‐associated epilepsy (AAE) 7.27 (3.74–11.95) and acute symptomatic seizures secondary to autoimmune encephalitis (ASSAE) 2.55 (0.69–5.60). From 1996 to 2019, the standardized incidence was 0.50 (0.29–0.77) per 100,000 person‐years overall, with AAE 0.36 (0.19–0.59) and ASSAE 0.15 (0.05–0.30). By period, the standardized incidence of all autoimmune seizures and epilepsies suggested a rising trend from 0.28 (95% CI, 0.06–0.68) in 1996 to 2003 to 0.60 (95% CI, 0.24–1.12) in 2004–2011, then plateaued at 0.53 (95% CI, 0.21–1.00) in 2012 to 2019 (rate ratio per 8‐year interval 1.30, 95% CI, 0.71–2.38), largely driven by the seropositive subgroup. The standardized DALY rate was 26.27 (95% Uncertainly intervals [UI], 5.30–67.09) per 100,000 (YLL 20.07 [0.00–60.21] and YLD 6.20 [5.06–7.39]). Interpretation Although autoimmune seizures and epilepsies remain rare, they result in a disproportionately high disease burden. ANN NEUROL 2026;100:74–83