CPVT-associated calmodulin variants N53I and A102V dysregulate Ca2+ signalling via different mechanisms

Area of Science:

• Molecular Cardiology
• Ion Channel Regulation
• Genetic Basis of Arrhythmias

Background:

• Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a life-threatening inherited arrhythmia.
• Mutations in calmodulin (CaM), a calcium sensor, are implicated in CPVT, but the precise molecular mechanisms are not fully understood.
• CaM interacts with the cardiac ryanodine receptor (RyR2), a key regulator of calcium release in cardiomyocytes.

Purpose of the Study:

• To investigate the molecular mechanisms underlying CPVT by examining the effects of CPVT-associated CaM variants (N53I and A102V) on CaM structure and function.
• To elucidate how these CaM variants impact Ca2+ fluxes and RyR2 interaction in live cells.

Main Methods:

• Co-expression of wild-type (WT) and mutant CaM (N53I, A102V) with RyR2 in HEK293 cells.
• High-resolution crystal structure determination of CaM variants bound to the RyR2 binding domain.
• Measurement of Ca2+ fluxes and oscillations in live cells.
• Assessment of CaMKIIδ (CAMK2D) phosphorylation activity.

Main Results:

• Both Ca2+/CaM-N53I and Ca2+/CaM-A102V showed reduced interaction with the RyR2 binding domain.
• Crystal structures revealed subtle conformational changes in CaM-N53I, while CaM-A102V structure was similar to WT.
• Co-expression of CPVT-associated CaM variants with RyR2 significantly prolonged Ca2+ event duration.
• CaM-A102V exhibited a lower frequency of Ca2+ oscillations and increased CaMKIIδ phosphorylation activity compared to CaM-WT.

Conclusions:

• CPVT-associated CaM variants N53I and A102V impair CaM's interaction with RyR2, leading to altered Ca2+ handling.
• These molecular defects contribute to the arrhythmogenic substrate in CPVT.
• Findings provide critical insights for developing targeted therapeutic strategies for CPVT.