Calmodulin mutations causing catecholaminergic polymorphic ventricular tachycardia confer opposing functional and biophysical molecular changes

Abstract

Calmodulin (CaM) is the central mediator of intracellular Ca2+ signalling in cardiomyocytes, where it conveys the intricate Ca 2+ transients to the proteins controlling cardiac contraction. We recently linked two separate mutations in CaM (N53I and N97S) to dominantly inherited catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmic disorder in which exercise or acute emotion can lead to syncope and sudden cardiac death. Given the ubiquitous presence of CaM in all eukaryote cells, it is particular intriguing that carriers of either mutation show no additional symptoms. Here, we investigated the effects of the CaM CPVT mutations in a zebrafish animal model. Three-day-old embryos injected with either CaM mRNA showed no detectable pathologies or developmental abnormalities. However, embryos injected with CPVT CaM mRNA displayed increased heart rate compared to wild-type CaM mRNA under b-adrenergic stimulation, demonstrating a conserved dominant cardiac specific effect between zebrafish and human carriers of these mutations. Motivated by the highly similar physiological phenotypes, we compared the effects of the N53I and N97S mutations on the biophysical and functional properties of CaM. Surprisingly, the mutations have opposing effects on CaM C-lobe Ca 2+ binding affinity and kinetics, and changes to the CaM N-lobe Ca 2+ binding are minor and specific to the N53I mutation. Furthermore, both mutations induce differential perturbations to structure and stability towards unfolding. Our results suggest different molecular disease mechanisms for the CPVT (N53I and N97S mutations) and strongly support that cardiac contraction is the physiological process most sensitive to CaM integrity.

Distinctive malfunctions of calmodulin mutations associated with heart RyR2-mediated arrhythmic disease

“…9,12,18,38 Using CD spectroscopy, in our study and others, there are no gross changes observed in tertiary structure, either in the absence or presence of Ca 2+ . 12,38 Decrease in thermal stability compared to WT is, however, a feature of most of the studied CaM mutations. 9,38 None of the CaM mutations in our, or any other, study affected Ca 2+ binding to the N-lobe.…”

“…12,38 Decrease in thermal stability compared to WT is, however, a feature of most of the studied CaM mutations. 9,38 None of the CaM mutations in our, or any other, study affected Ca 2+ binding to the N-lobe. However, nearly all the CaM mutations resulted in decreased Ca 2+ binding in the C-lobe to varying degrees 8,9,10,17,18,38 with the exception of N54I 8,17,38 which, perhaps unsurprisingly, is located in the loop between EF-hands 1 and 2 of the N-lobe ( Figure 5).…”

“…Thus, there was no apparent effect on RyR2 activity to suggest the potential for F142L to generate a CPVT phenotype. 8,9,12,18,38 leading to different Ca 2+ binding affinities and/or changes in the interaction with the apo-CaM or Ca 2+ -CaM binding sites on a variety of vulnerable ion channel complexes. 42 Any of these aspects could contribute to which ion channel type will be most severely affected.…”

Calcium triggers reversal of calmodulin on nested anti-parallel sites in the IQ motif of the neuronal voltage-dependent sodium channel Na V 1.2

“…[111] Exome analyses of humans have identified calmodulinopathies that are also specific to one domain of CaM. [112–115] Some of the identified positions, such as D95 in site III, have been found to be deleterious in both Paramecium and human studies, suggesting that their mechanisms of action are conserved.…”

Endogenous Natural Complement Inhibitor Regulates Cardiac Development

“…Videos were recorded for 20 (or 4) s at a frame rate of 100 per second. To determine heart rates, an algorithm was written in MATLAB (MathWorks, Natick, MA), as described previously (31). The supplemental videos were acquired using a Zeiss Axio Observer.Z1 microscope equipped with an AxioCam HRm camera (frame rate of five per second).…”