Engineering an Anti-Arrhythmic Calmodulin

Abstract

The cardiac ryanodine receptor (RYR2) is a critical Ca²+ handling protein. Several post-translational modifications, mutations, and altered interactions between RYR2 and its regulatory proteins can all increase the RYR2 open probability (P0), disrupting normal Ca²+ handling, leading to arrhythmia. There is a great need for therapeutic strategies that decrease RYR2 P0. Normally, calmodulin (CaM) decreases RYR2 P0, decreasing Ca²+ sparks and waves. Naturally occurring mutations in CaM have recently been identified that instead increase RYR2 P0 and cause arrhythmia. Using these CaM mutations as tools, we have discovered that the N-terminal rate of Ca²+ dissociation from the CaM-RYR2 complex is an important factor that modulates RYR2 inhibition by CaM. That is, the longer Ca²+ remains bound to the CaM-RYR2 complex the longer RYR2 remains refractory, ultimately influencing the frequency of cardiac myocyte sparks and waves. We hypothesize that slowing the N-terminal Ca²+ dissociation rate from CaM bound to RYR2 will decrease RYR2 P0 and prevent arrhythmias. Using both rational engineering (GSH M37Q CaM) and nature's design principles (soybean CaM4) to sensitize CaM to Ca²+, we are attempting to create therapeutic CaMs against defective RYR2 regulation. Excitingly, our therapeutic GSH M37Q CaM decreases the arrhythmogenic potential of myocytes isolated from the pro-arrhythmic calsequestrin knockout mouse. Thus, tuning CaM's regulation of RYR2 through Ca²+ dissociation is a viable therapeutic strategy to prevent arrhythmia.