This Tuesday 30/03/2023 at 10:00 a.m., Mr. David Carreras has completed the reading of the doctoral thesis entitled: “Study of intracellular calcium release in iPSC-derived cardiomyocytes from patients with catecholaminergic polymorphic ventricular tachycardia (CPVT)." directed by Dr. Guillermo J. Pérez and Dr. Ramon Brugada.
In a small region of the island of Gran Canaria, 11 cases of sudden cardiac death (SCD) were registered in emotional or physical stress situations in young individuals between 1994 and 2007 in four seemingly unrelated families. An extensive genealogical study allowed to connect the four families within a large family tree of more than 2000 members with a common ancestor born in 1749. Genetic studies identified a mutation in the type 2 ryanodine receptor (RYR2) gene RYR2_c.G1069A (RYR2_p.G357S) that was associated to the SCD cases in the family. The RYR2 gene is the main gene linked to lethal arrhythmia called catecholaminergic polymorphic ventricular tachycardia (CPVT). Initially, 179 living carriers of this mutation and 36 dead individuals were identified. Among the dead individuals, 6 were genotyped positive and the others had died under highly suggestive documented circumstances of CPVT.
Studies performed with heterologous expression models, in which the expression of the mutated protein is induced, showed that the mutation promoted a gain of function in the activity of RYR2 under mimicked stress situations. Subsequent studies proposed that the variability in the penetrance and expressivity of the phenotype in the family could be explained by the decrease in the RYR2 protein amount caused by the mutation. The generation of cardiomyocytes from induced pluripotent stem cells (iPSC-CM) of individuals carrying the mutation, gave us the opportunity to determine the pathogenic effects of the mutation in a cellular relevant model, and how the individual genetic background could influence the phenotype of the disease.
Our study included one healthy control and five RYR2_p.G357S mutation carriers from three family nuclei: I) Two heterozygous siblings: one symptomatic (presyncope at age 19 and SCD recovered at age 21) and his asymptomatic sister. II) Two individuals with a second-degree kinship, who were first (syncope at the age of 39) and third generation (syncope at 8 years of age). III) An individual, homozygous for the mutation, without direct kinship with other individuals (syncope at 8 years of age). All the mutation carriers have an implanted automatic defibrillator except for the asymptomatic individual of the first family nucleus.
Functional experiments performed with the iPSC-CM of the individuals included in the study, using caffeine as a specific activator of RYR2, showed a gain of function in channel activity under basal conditions, compared to the healthy control. This enhanced RYR2 activity could not be further increased by isoproterenol stimulation. Moreover, we observed a decreased function in the case of iPSC-CM derived from second-degree relatives. At the molecular level, we were able to determine that protein and RNA levels of RYR2 were similar to the healthy control. This showed that the low penetrance of the mutation was not caused by a decrease in the amount of RYR2.
Our results indicate that the RYR2_p.G357S mutation promotes both a gain of function under basal conditions, and an abnormal response to β-adrenergic stimulation with isoproterenol. This could provide the cell substrate necessary for the generation of malignant arrhythmias. The reduced penetrance and the variable expressivity of the mutation in the large family of the island of Gran Canaria could not be attributed to the degradation of RYR2. We consider instead that the individual genetic variants found in the genome of mutation carrier donors could increase or decrease the severity of the phenotype.