Generation and characterization of a mouse model harboring the exon-3 deletion in the cardiac ryanodine receptor

Area of Science:

• Cardiovascular Genetics
• Molecular Cardiology
• Genetic Disease Mechanisms

Background:

• A large genomic deletion in the cardiac ryanodine receptor (RYR2) gene, specifically an in-frame deletion of exon-3 (Ex3-del), is linked to various clinical phenotypes, including catecholaminergic polymorphic ventricular tachycardia (CPVT).
• Understanding the precise disease mechanism of this RYR2 Ex3-del mutation is crucial for explaining patient variability.

Purpose of the Study:

• To investigate the functional consequences of the RYR2 exon-3 deletion (Ex3-del) mutation.
• To elucidate the underlying disease mechanism of Ex3-del-associated cardiac arrhythmias using a mouse model.

Main Methods:

• Generation of a mouse model with a targeted deletion of RyR2 exon-3 and flanking intronic sequences.
• Phenotypic analysis of heterozygous Ex3-del mice (Ex3-del+/-), including assessment of susceptibility to CPVT and characterization of cardiomyocyte calcium handling.
• Evaluation of RyR2 protein expression levels via immunoblotting and investigation of cardiac function following conditional knockout of the wild-type RyR2 allele.

Main Results:

• Heterozygous Ex3-del mice survived but did not exhibit susceptibility to CPVT.
• Cardiomyocytes from Ex3-del+/- mice showed altered calcium transient dynamics despite similar amplitudes compared to wild-type cells.
• Significantly reduced RyR2 protein expression was observed in Ex3-del+/- mutant hearts, suggesting the Ex3-del allele impacts protein stability or expression.
• Conditional knockout of the wild-type RyR2 allele in Ex3-del+/- mice resulted in bradycardia and lethality.

Conclusions:

• The absence of CPVT in Ex3-del+/- mice is likely due to the markedly reduced expression of the mutant RyR2 allele, allowing predominant expression of the wild-type allele.
• The Ex3-del mutation significantly impacts RyR2 protein expression levels in the mouse heart.
• The findings provide insights into the relationship between RyR2 Ex3-del mutation, protein expression, and the observed phenotypic variability in human patients.