Recent work has identified missense mutations in calmodulin (CaM) that are associated with severe early-onset long-QT syndrome (LQTS), leading to the proposition that altered CaM function may contribute to the molecular etiology of this subset of LQTS. To date, however, no experimental evidence has established these mutations as directly causative of LQTS substrates, nor have the molecular targets of CaM mutants been identified. Here, therefore, we test whether expression of CaM mutants in adult guinea-pig ventricular myocytes (aGPVM) induces action-potential prolongation, and whether affiliated alterations in the Ca2+ regulation of L-type Ca2+ channels (LTCC) might contribute to such prolongation. In particular, we first overexpressed CaM mutants in aGPVMs, and observed both increased action potential duration (APD) and heightened Ca2+ transients. Next, we demonstrated that all LQTS CaM mutants have the potential to strongly suppress Ca2+/CaM-dependent inactivation (CDI) of LTCCs, whether channels were heterologously expressed in HEK293 cells, or present in native form within myocytes. This attenuation of CDI is predicted to promote action-potential prolongation and boost Ca2+ influx. Finally, we demonstrated how a small fraction of LQTS CaM mutants (as in heterozygous patients) would nonetheless suffice to substantially diminish CDI, and derange electrical and Ca2+ profiles. In all, these results highlight LTCCs as a molecular locus for understanding and treating CaM-related LQTS in this group of patients.
Rationale Calmodulinopathies comprise a new category of potentially life-threatening genetic arrhythmia syndromes capable of producing severe long QT syndrome (LQTS) with mutations involving either CALM1, CALM2, or CALM3. The underlying basis of this form of LQTS is a disruption of Ca2+/CaM-dependent inactivation (CDI) of L-type Ca2+ channels (LTCCs). Objective To gain insight into the mechanistic underpinnings of calmodulinopathies and devise new therapeutic strategies for the treatment of this form of LQTS. Methods and Results We generated and characterized the functional properties of iPSC-derived cardiomyocytes (iPSC-CMs) from a patient with D130G-CALM2-mediated LQTS, thus creating a platform with which to devise and test novel therapeutic strategies. The patient-derived iPSC-CMs display (1) significantly prolonged action potentials (APs), (2) disrupted Ca2+ cycling properties, and (3) diminished CDI of LTCCs. Next, taking advantage of the fact that calmodulinopathy patients harbor a mutation in only one of six redundant CaM-encoding alleles, we devised a strategy using CRISPR interference (CRISPRi) to selectively suppress the mutant gene while sparing the wild-type counterparts. Indeed, suppression of CALM2 expression produced a functional rescue in iPSC-CMs with D130G-CALM2, as shown by the normalization of AP duration and CDI following treatment. Moreover, CRISPRi can be designed to achieve selective knockdown of any of the three CALM genes, making it a generalizable therapeutic strategy for any calmodulinopathy. Conclusions Overall, this therapeutic strategy holds great promise for calmodulinopathy patients as it represents a generalizable intervention capable of specifically altering CaM expression and potentially attenuating LQTS-triggered cardiac events, thus initiating a path towards precision medicine.
Voltage-gated Na and Ca2+ channels represent two major ion channel families that enable myriad biological functions including the generation of action potentials and the coupling of electrical and chemical signaling in cells. Calmodulin regulation (calmodulation) of these ion channels comprises a vital feedback mechanism with distinct physiological implications. Though long-sought, a shared understanding of the channel families remained elusive for two decades as the functional manifestations and the structural underpinnings of this modulation often appeared to diverge. Here, we review recent advancements in the understanding of calmodulation of Ca2+ and Na channels that suggest a remarkable similarity in their regulatory scheme. This interrelation between the two channel families now paves the way towards a unified mechanistic framework to understand vital calmodulin-dependent feedback and offers shared principles to approach related channelopathic diseases. An exciting era of synergistic study now looms.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.