Worawan B. Limpitikul

Calmodulin mutations associated with long QT syndrome prevent inactivation of cardiac L-type Ca2+ currents and promote proarrhythmic behavior in ventricular myocytes

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 Ca(2+) regulation of L-type Ca(2+) 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 Ca(2+) transients. Next, we demonstrated that all LQTS CaM mutants have the potential to strongly suppress Ca(2+)/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 Ca(2+) 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 Ca(2+) profiles. In all, these results highlight LTCCs as a molecular locus for understanding and treating CaM-related LQTS in this group of patients.

A Precision Medicine Approach to the Rescue of Function on Malignant Calmodulinopathic Long QT Syndrome

Rationale: Calmodulinopathies comprise a new category of potentially life-threatening genetic arrhythmia syndromes capable of producing severe long-QT syndrome (LQTS) with mutations involving CALM1, CALM2, or CALM3. The underlying basis of this form of LQTS is a disruption of Ca2+/calmodulin (CaM)-dependent inactivation of L-type Ca2+ channels. 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 induced pluripotent stem cell–derived cardiomyocytes from a patient with D130G-CALM2–mediated LQTS, thus creating a platform with which to devise and test novel therapeutic strategies. The patient-derived induced pluripotent stem cell–derived cardiomyocytes display (1) significantly prolonged action potentials, (2) disrupted Ca2+ cycling properties, and (3) diminished Ca2+/CaM-dependent inactivation of L-type Ca2+ channels. Next, taking advantage of the fact that calmodulinopathy patients harbor a mutation in only 1 of 6 redundant CaM-encoding alleles, we devised a strategy using CRISPR interference to selectively suppress the mutant gene while sparing the wild-type counterparts. Indeed, suppression of CALM2 expression produced a functional rescue in induced pluripotent stem cell–derived cardiomyocytes with D130G-CALM2, as shown by the normalization of action potential duration and Ca2+/CaM-dependent inactivation after treatment. Moreover, CRISPR interference can be designed to achieve selective knockdown of any of the 3 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 toward precision medicine.

Towards a Unified Theory of Calmodulin Regulation (Calmodulation) of Voltage-Gated Calcium and Sodium Channels

Voltage-gated Na and Ca(2+) 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 Ca(2+) 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.

Influence of Electromechanical Activity on Cardiac Differentiation of Mouse Embryonic Stem Cells

During differentiation, mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) receive electromechanical cues from spontaneous beating. Therefore, promoting electromechanical activity via electrical pacing or suppressing it by drug treatment might affect the cellular functional development. Electrical pacing was applied to confluent monolayers of mESC-CMs during late-stage differentiation (days 16–18). Alternatively, spontaneous contraction was suppressed by (a) blocking ion currents with CsCl (HCN channel), trazodone (T-type Ca2+ channel), or both CsCl and trazodone on days 11–18; or (b) applying blebbistatin (excitation–contraction uncoupler) on days 11–14. Electrophysiological properties and gene expression were examined on day 19 and 18, respectively. Optical mapping revealed no significant difference in conduction velocity (CV) in paced vs. non-paced monolayers, nor were there significant changes in gene expression of connexin-43, Na–Ca exchanger (NCX), or myosin heavy chain (MHC). However, CV variability among differentiation batches and CV heterogeneity within individual monolayers were significantly lower in paced mESC-CMs. Alternatively, while the four drug treatments suppressed contraction with varying degrees (up to complete inhibition), there was no significant difference in CV for any of the treatments compared with controls. Trazodone treatment significantly reduced CV variability as compared to controls, whereas CsCl treatment significantly reduced CV heterogeneity. Distinct changes in gene expression of connexin-43, MHC, HCNl, Cav3.1/3.2 were not observed. Electrical pacing, but not suppression of spontaneous contraction, during late-stage differentiation reduces the intrinsic variability of CV among differentiation batches and across individual monolayers, which can be beneficial in the application of ESCs for myocardial tissue repair.

An autism-associated mutation in CaV1.3 channels has opposing effects on voltage- and Ca2+-dependent regulation

CaV1.3 channels are a major class of L-type Ca2+ channels which contribute to the rhythmicity of the heart and brain. In the brain, these channels are vital for excitation-transcription coupling, synaptic plasticity, and neuronal firing. Moreover, disruption of CaV1.3 function has been associated with several neurological disorders. Here, we focus on the de novo missense mutation A760G which has been linked to autism spectrum disorder (ASD). To explore the role of this mutation in ASD pathogenesis, we examined the effects of A760G on CaV1.3 channel gating and regulation. Introduction of the mutation severely diminished the Ca2+-dependent inactivation (CDI) of CaV1.3 channels, an important feedback system required for Ca2+ homeostasis. This reduction in CDI was observed in two major channel splice variants, though to different extents. Using an allosteric model of channel gating, we found that the underlying mechanism of CDI reduction is likely due to enhanced channel opening within the Ca2+-inactivated mode. Remarkably, the A760G mutation also caused an opposite increase in voltage-dependent inactivation (VDI), resulting in a multifaceted mechanism underlying ASD. When combined, these regulatory deficits appear to increase the intracellular Ca2+ concentration, thus potentially disrupting neuronal development and synapse formation, ultimately leading to ASD.

A rendezvous with the queen of ion channels: Three decades of ion channel research by David T Yue and his Calcium Signals Laboratory

David T. Yue was a renowned biophysicist who dedicated his life to the study of Ca2+ signaling in cells. In the wake of his passing, we are left not only with a feeling of great loss, but with a tremendous and impactful body of work contributed by a remarkable man. Davids research spanned the spectrum from atomic structure to organ systems, with a quantitative rigor aimed at understanding the fundamental mechanisms underlying biological function. Along the way he developed new tools and approaches, enabling not only his own research but that of his contemporaries and those who will come after him. While we cannot hope to replicate the eloquence and style we are accustomed to in Davids writing, we nonetheless undertake a review of Davids chosen field of study with a focus on many of his contributions to the calcium channel field.

Conservation of cardiac L-type Ca2+ channels and their regulation in Drosophila: A novel genetically-pliable channelopathic model

Dysregulation of L-type Ca2+ channels (LTCCs) underlies numerous cardiac pathologies. Understanding their modulation with high fidelity relies on investigating LTCCs in their native environment with intact interacting proteins. Such studies benefit from genetic manipulation of endogenous channels in cardiomyocytes, which often proves cumbersome in mammalian models. Drosophila melanogaster, however, offers a potentially efficient alternative as it possesses a relatively simple heart, is genetically pliable, and expresses well-conserved genes. Fluorescence in situ hybridization confirmed an abundance of Ca-α1D and Ca-α1T mRNA in fly myocardium, which encode subunits that specify hetero-oligomeric channels homologous to mammalian LTCCs and T-type Ca2+ channels, respectively. Cardiac-specific knockdown of Ca-α1D via interfering RNA abolished cardiac contraction, suggesting Ca-α1D (i.e. A1D) represents the primary functioning Ca2+ channel in Drosophila hearts. Moreover, we successfully isolated viable single cardiomyocytes and recorded Ca2+ currents via patch clamping, a feat never before accomplished with the fly model. The profile of Ca2+ currents recorded in individual cells when Ca2+ channels were hypomorphic, absent, or under selective LTCC blockage by nifedipine, additionally confirmed the predominance of A1D current across all activation voltages. T-type current, activated at more negative voltages, was also detected. Lastly, A1D channels displayed Ca2+-dependent inactivation, a critical negative feedback mechanism of LTCCs, and the current through them was augmented by forskolin, an activator of the protein kinase A pathway. In sum, the Drosophila heart possesses a conserved compendium of Ca2+ channels, suggesting that the fly may serve as a robust and effective platform for studying cardiac channelopathies.