Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by a single point mutation in the cardiac type 2 ryanodine receptor (RyR2). Using a knockin (KI) mouse model (R2474S/+), we previously reported that a single point mutation within the RyR2 sensitizes the channel to agonists, primarily mediated by defective interdomain interaction within the RyR2 and subsequent dissociation of calmodulin (CaM) from the RyR2. Here, we examined whether CPVT can be genetically rescued by enhancing the binding affinity of CaM to the RyR2. We first determined whether there is a possible amino acid substitution within the CaM-binding domain in the RyR2 (3584–3603 residues) that can enhance its binding affinity to CaM and found that V3599K substitution showed the highest binding affinity of CaM to the CaM-binding domain. Hence, we generated a heterozygous KI mouse model (V3599K/+) with a single amino acid substitution in the CaM-binding domain of the RyR2 and crossbred it with the heterozygous CPVT-associated R2474S/+-KI mouse to obtain a double-heterozygous R2474S/V3599K-KI mouse model. The CPVT phenotypes — bidirectional or polymorphic ventricular tachycardia, spontaneous Ca ²⁺ transients, and Ca ²⁺ sparks — were all inhibited in the R2474S/V3599K mice. Thus, enhancement of the CaM-binding affinity of the RyR2 is essential to prevent CPVT-associated arrhythmogenesis.

The type 2 ryanodine receptor (RyR2) is a giant Ca²⁺ (Ca)-releasing channel on the sarcoplasmic reticulum (SR) membrane, with subunits composed of 5000 amino acids constituting a homotetrameric channel. The N-terminal (1-220) and central (2300-2500) domain interactions (inter-subunit zipping interfaces) within RyR2 are located in close proximity to each other between different neighboring subunits and play an important "cornerstone" role in maintaining the tetrameric structure of RyR2. External stress such as oxidative stress causes Ca leak by destabilizing RyR2 (instability of the tetrameric structure) due to domain unzipping between N-terminal (1-220) and central (2300-2500) domains, followed by dissociation of calmodulin (CaM: binds to the RyR2 and stabilize RyR2) from RyR2. Ca leak from SR causes arrhythmias and myocardial dysfunction. RyR2 is also present in the endoplasmic reticulum (ER), thus it is not surprising that undesired Ca release from RyR2 on the ER is closely associated with various diseases involving ER dysfunction such as neurodegenerative diseases, diabetes, metabolic dysfunction-associated steatotic liver disease, chronic kidney disease, and autoimmune diseases. Pharmacological or genetic (point mutations within RyR2 that increase CaM-RyR2 affinity: knock-in V3599K) RyR2 structural stabilization has shown potential therapeutic effects not only for SR failure-related diseases (malignant hyperthermia, arrhythmia, heart failure) but also for ER failure-related disease. RyR2-stabilizers may function as a panacea for aging-related diseases.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited stress‐induced arrhythmogenic disease often caused by point variants in the cardiac ryanodine receptor (RyR2) that enhance diastolic sarcoplasmic reticulum (SR) Ca²⁺ leak. These RyR2 variants cluster in three hot‐spots (N‐terminal, central and C‐terminal domains). We previously demonstrated a pathologic diastolic RyR2 conformation in heart failure, oxidative stress and CaMKII phosphorylation exhibiting a trilogy of effects: (1) reduced calmodulin (CaM)‐RyR2 affinity, (2) enhanced unzipping peptide (DPc10) binding and (3) elevated diastolic SR Ca²⁺ leak that are dantrolene‐sensitive. Here we test whether this pathological trilogy occurs in CPVT knock‐in (KI) mice bearing N‐terminal (R176Q/+), central (R2474S/+) and C‐terminal variants (R4496C/+). Isolated saponin‐permeabilized ventricular myocytes from all three KI mice exhibited unaltered baseline CaM and DPc10 binding to RyR2 versus wild‐type (WT) myocytes. However cAMP‐induced protein kinase A (PKA)‐dependent RyR2 phosphorylation revealed the pathological trilogy, but only in R176Q and R2474S KI myocytes. That contrasts with WT and R4496C KI myocytes where cAMP enhanced SR Ca²⁺ leak without altering either CaM‐ or DPc10‐RyR2 affinity. We conclude that CPVT‐linked RyR2 variants at baseline may not exhibit the pathological diastolic trilogy above, but that trilogy is induced by PKA activation only in N‐terminal and central domain CPVT variants. Thus the mechanism of arrhythmogenic RyR2 leak in pore domain variants (e.g. R4496C) may not involve the same pathological conformational changes as in N‐terminal and central domain variants. This may inform which specific CPVT‐linked RyR2 variants may benefit from therapeutic strategies that target this pathological trilogy conformation. image Key points: We tested whether CPVT‐linked RyR2 variants in the N‐terminal (R176Q/+), central (R2474S/+) and C‐terminal/pore (R4496C/+) domains mimic a pathologic trilogy of reduced CaM affinity, enhanced DPc10 peptide binding and arrhythmogenic leaky RyR2. At baseline, none of these RyR2 variant knock‐in ventricular myocytes exhibited the above trilogy that is seen with heart failure, oxidative stress and CaMKII activation. However PKA activation by cAMP promotes that pathological trilogy, but only in the N‐terminal and central domain variant myocytes, not the pore domain variant. Targeted therapies may need to be different for different CPVT‐linked variants.

Background: Hydrogen gas (H2) has been shown to be effective in the treatment of various clinical conditions, from acute illnesses to chronic illnesses. However, its clinical indications and the corresponding appropriate hydrogen delivery methods have yet to be determined. This is due to the fact that the pharmacokinetics and pharmacodynamics of hydrogen in each delivery method have not been experimentally proven. Here, we verified the pharmacokinetics of hydrogen after the infusion of hydrogen-saturated saline. Methods: Hydrogen-saturated saline was prepared and checked for sterility and component specifications. Hydrogen-saturated saline was administered intravenously (125 mL/h) through the left internal jugular vein of pigs, and the blood hydrogen concentration was measured over time. Results: It was confirmed that hydrogen can be safely mixed under pressure into intravenous solutions (pharmaceutical products) without the contamination of foreign substances by using a needle-less vial access cannula. No change in the PH or composition of the solution was observed due to hydrogen filling. The hydrogen concentrations of blood samples collected from the left internal jugular vein 3 cm to the heart from the tip of the infusion line were 6.4 (30 min), 4.7 (60 min), 4.9 (90 min), and 5.3 (120 min) ppb w/w, respectively. The hydrogen concentrations of blood samples collected from the right atrium were 0.7 (30 min), 0.5 (60 min), 0.7 (90 min), and 0.7 (120 min) ppb, respectively. The hydrogen concentration of blood samples collected from the right internal carotid artery were 0.1 (pre), 0.2 (30 min), 0.3 (60 min), 0.0 (90 min), and 0.0 (120 min) ppb w/w, respectively. Conclusions: We confirmed that hydrogen could be safely pressurized and filled into intravenous (pharmaceutical) solution without contamination by foreign objects using a needle-free vial access cannula. When saturated hydrogen saline was dripped intravenously, almost all of the hydrogen was expelled during its passage through the lungs and could not be supplied to the arterial side.