L-type Calcium Channel Function Research Articles

Natural L-type calcium channels antagonists from Chinese medicine

L-type calcium channels (LTCCs), the largest subfamily of voltage-gated calcium channels (VGCCs), are the main channels for Ca2+ influx during extracellular excitation. LTCCs are widely present in excitable cells, especially cardiac and cardiovascular smooth muscle cells, and participate in various Ca2+-dependent processes. LTCCs have been considered as worthy drug target for cardiovascular, neurological and psychological diseases for decades. Natural products from Traditional Chinese medicine (TCM) have shown the potential as new drugs for the treatment of LTCCs related diseases. In this review, the basic structure, function of LTCCs, and the related human diseases caused by structural or functional abnormalities of LTCCs, and the natural LTCCs antagonist and their potential usages were summarized.

Abstract P3142: Engineering A Genetically Encoded Enhancer Of L-type Ca 2+ Currents

L-type calcium channels (LTCC) play a crucial role as they serve as vital portals for Ca 2+ entry into cardiac myocytes, neurons, and other physiological systems. Altered LTCC function is linked to various pathologies including cardiac arrhythmias and neurodevelopmental disorders. In particular, genetic studies have identified loss-of-function mutations in Ca V 1.2 channels in patients with shortened QT interval and Brugada Syndrome, as well as neuropsychiatric disorders. Given the broad physiological importance of LTCC in various tissues, conventional pharmacological agonists of LTCC may yield off-target effects and toxicity. Therefore, new approaches to upregulate LTCC function in a cell-type dependent manner is sought after. To do so, we here develop a genetically encoded enhancer of calcium channels (GeeCC, “geek”) by leveraging both a cardiac specific L-type channel modulator, leucine rich repeat containing protein 10 (Lrrc10), and selective nanobodies that target the LTCC complex. Specifically, Lrrc10 co-expression markedly upregulates LTCC currents. Furthermore, mutations in Lrrc10 are linked to cardiac diseases, including cardiomyopathies. Through extensive structure-function analysis, we identified a minimal action domain within Lrrc10 that is critical for functional modulation of LTCC. However, as this segment alone has a low affinity for the channel complex, its expression alone yielded minimal changes in Ca 2+ current amplitude. However, fusion of this Lrrc10 domain to a nanobody (nb.F3) that targets the Ca 2+ channel beta subunit resulted in a significant increase in the LTCC current amplitude. In depth analysis revealed exquisite selectivity of GeeCC for Ca V 1.2/Ca V 1.3 channels. In mouse cardiomyocytes, deployment of GeeCC via adenovirus enhances endogenous L-type currents when compared to uninfected controls or cardiomyocytes infected with nanobodies alone. Additionally, in neurons, co-expression of GeeCC enhanced excitation-transcription coupling. In all, we have developed a genetically-encoded approach to selectively enhance L-type currents. This strategy may enable new insights into cardiovascular and neuronal physiology and pathophysiology, and may have potential therapeutic applications.

Aging impairs the clustering and reduces the activity of the L-type calcium channels in the heart's pacemaker

Every heartbeat is initiated by a spontaneous electrical signal generated inside the cardiac pacemaker. In mammals, including humans, aging leads to the slowdown of the pacemaker rate, which can lead to pathological dysfunction and death. However, the mechanisms behind the age-associated slowdown of the pacemaker are not well understood. The automaticity of the pacemaker relies on a finely tuned balance of ionic currents, where L-type calcium channels play a central role. CaV1.2 and CaV1.3 channels are key players involved in the diastolic depolarization phase and action potential upstroke. In this project, we studied the effects of aging on CaV1.2 and CaV1.3 calcium channel function and regulation. Here we recorded the pacemaker activity and calcium currents on isolated spindle pacemaker cells with patch clamp. The clustering of LTCC has been highlighted with Super-resolution microscopy (GSD). We found that in mouse pacemaker cells, aging causes a reduction of calcium entry through L-type calcium channels, which is not accompanied by a reduction of protein expression. In turn, we found that aging disrupts calcium channel function at different levels. First, aging reduces the expression of L-type calcium channels at the plasma membrane. Second, aging reduces the clustering and nano-organization of L-type calcium channels. And finally, aging reduces the open channel probability, a sign of decreased functional coupling. Moreover, aging also alters the association between L-type calcium channels and beta-adrenergic receptors, altering the adrenergic modulation of the pacemaker cells. Next, we tested the hypothesis that reduced open probability of L-type calcium channels was responsible for a slower firing frequency. Interestingly, we found that the application of BayK was enough to restore intrinsic pacemaker rates in old cells to those levels observed in young cells. Overall, our findings provide evidence that the proper organization and function of L-type calcium channels is affected by aging and that this dysfunction contributes to the slowdown of pacemaker cells in old animals.

Novel Calmodulin Variant p.E46K Associated With Severe Catecholaminergic Polymorphic Ventricular Tachycardia Produces Robust Arrhythmogenicity in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

CaM (calmodulin) is a ubiquitously expressed, multifunctional Ca2+ sensor protein that regulates numerous proteins. Recently, CaM missense variants have been identified in patients with malignant inherited arrhythmias, such as long QT syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the exact mechanism of CaM-related CPVT in human cardiomyocytes remains unclear. In this study, we sought to investigate the arrhythmogenic mechanism of CPVT caused by a novel variant using human induced pluripotent stem cell (iPSC) models and biochemical assays. We generated iPSCs from a patient with CPVT bearing CALM2 p.E46K. As comparisons, we used 2 control lines including an isogenic line, and another iPSC line from a patient with long QT syndrome bearing CALM2 p.N98S (also reported in CPVT). Electrophysiological properties were investigated using iPSC-cardiomyocytes. We further examined the RyR2 (ryanodine receptor 2) and Ca2+ affinities of CaM using recombinant proteins. We identified a novel de novo heterozygous variant, CALM2 p.E46K, in 2 unrelated patients with CPVT accompanied by neurodevelopmental disorders. The E46K-cardiomyocytes exhibited more frequent abnormal electrical excitations and Ca2+ waves than the other lines in association with increased Ca2+ leakage from the sarcoplasmic reticulum via RyR2. Furthermore, the [3H]ryanodine binding assay revealed that E46K-CaM facilitated RyR2 function especially by activating at low [Ca2+] levels. The real-time CaM-RyR2 binding analysis demonstrated that E46K-CaM had a 10-fold increased RyR2 binding affinity compared with wild-type CaM which may account for the dominant effect of the mutant CaM. Additionally, the E46K-CaM did not affect CaM-Ca2+ binding or L-type calcium channel function. Finally, antiarrhythmic agents, nadolol and flecainide, suppressed abnormal Ca2+ waves in E46K-cardiomyocytes. We, for the first time, established a CaM-related CPVT iPSC-CM model which recapitulated severe arrhythmogenic features resulting from E46K-CaM dominantly binding and facilitating RyR2. In addition, the findings in iPSC-based drug testing will contribute to precision medicine.

Aging differentially affects LTCC function in hippocampal CA1 and piriform cortex pyramidal neurons.

Aging is associated with cognitive decline and memory loss in humans. In rats, aging-associated neuronal excitability changes and impairments in learning have been extensively studied in the hippocampus. Here, we investigated the roles of L-type calcium channels (LTCCs) in the rat piriform cortex (PC), in comparison with those of the hippocampus. We employed spatial and olfactory tasks that involve the hippocampus and PC. LTCC blocker nimodipine administration impaired spontaneous location recognition in adult rats (6-9months). However, the same blocker rescued the spatial learning deficiency in aged rats (19-23months). In an odor-associative learning task, infusions of nimodipine into either the PC or dorsal CA1 impaired the ability of adult rats to learn a positive odor association. Again, in contrast, nimodipine rescued odor associative learning in aged rats. Aged CA1 neurons had higher somatic expression of LTCC Cav1.2 subunits, exhibited larger afterhyperpolarization (AHP) and lower excitability compared with adult neurons. In contrast, PC neurons from aged rats showed higher excitability and no difference in AHP. Cav1.2 expression was similar in adult and aged PC somata, but relatively higher in PSD95- puncta in aged dendrites. Our data suggest unique features of aging-associated changes in LTCCs in the PC and hippocampus.

Dissecting the molecular basis of human interneuron migration in forebrain assembloids from Timothy syndrome.

Defects in interneuron migration can disrupt the assembly of cortical circuits and lead to neuropsychiatric disease. Using forebrain assembloids derived by integration of cortical and ventral forebrain organoids, we have previously discovered a cortical interneuron migration defect in Timothy syndrome (TS), a severe neurodevelopmental disease caused by a mutation in the L-type calcium channel (LTCC) Cav1.2. Here, we find that acute pharmacological modulation of Cav1.2 can regulate the saltation length, but not the frequency, of interneuron migration in TS. Interestingly, the defect in saltation length is related to aberrant actomyosin and myosin light chain (MLC) phosphorylation, while the defect in saltation frequency is driven by enhanced γ-aminobutyric acid (GABA) sensitivity and can be restored by GABA-A receptor antagonism. Finally, we describe hypersynchronous hCS network activity in TS that is exacerbated by interneuron migration. Taken together, these studies reveal a complex role of LTCC function in human cortical interneuron migration and strategies to restore deficits in the context of disease.

Regulation of localization and function of L-type calcium channels by junctophilins

Striated muscle L-type calcium channels (LTCC) are localized specifically to the junctional membrane (JM) where the sarcolemma is closely apposed to the sarcoplasmic reticulum. Although this allocation of LTCC is critical for efficient excitation-contraction coupling in striated muscles, its underlying molecular mechanism has not been clarified. Junctophilins (JPs) stabilize the structure of JM by bridging the sarcolemmal and SR membranes. In addition, immunoprecipitation and pull-down assay revealed that the proximal C-terminus of CaV1.1 subunits directly binds to both JP1 and JP2, indicating that JPs might also directly recruit and hold LTCC in JM. Indeed, expression of a JP1 mutant lacking its C-terminus including the transmembrane domain in mouse skeletal muscles exerted a dominant-negative effect on endogenous JPs by impairing LTCC-RyR coupling at triads and reducing contractile force. To investigate a role of cardiac JP2 in a similar strategy, we injected adeno-associated virus vector expressing a C-terminus lacking JP2 mutant (JP2Δ427) driven by a cardiac troponin T promoter into C57BL/6 mice. Echocardiography recorded 4 weeks after the viral injection showed that the fractional shortening in JP2Δ427 group was significantly decreased compared to that of the control group. Calcium transient of isolated ventricular myocytes was significantly decreased by JP2Δ427 expression. Immunocytochemistry showed that JP2Δ427 recruited LTCC to the surface sarcolemma from T-tubules. Taken together, expression of C-terminus lacking JP mutants down-regulated contractile force by impairing ECC of skeletal and cardiac myocytes. Thus, the physical binding between LTCC and JP is essential for contraction of striated muscles.

Prenatal High-Sucrose Diet Induced Vascular Dysfunction in Thoracic Artery of Fetal Offspring.

Maternal nutrition during pregnancy is related to intrauterine fetal development. The authors' previous work reports that prenatal high sucrose (HS) diet impaired micro-vascular functions in postnatal offspring. It is unclear whether/how prenatal HS causes vascular injury during fetal life. Pregnant rats are fed with normal drinking water or 20% high-sucrose solution during the whole gestational period. Pregnant HS increases maternal weight before delivery. Fetal thoracic aorta is separated for experiments. Angiotensin II (AII)-stimulated vascular contraction of fetal thoracic arteries in HS group is greater, which mainly results from the enhanced AT1 receptor (AT1R) function and the downstream signaling. Nifedipine significantly increases vascular tension in HS group, indicating that the L-type calcium channels (LTCCs) function is strengthened. 2-Aminoethyl diphenylborinate (2-APB), inositol 1,4,5-trisphosphate receptors (IP3Rs) inhibitor, increases vascular tension induced by AII in HS group and ryanodine receptors-sensitive vascular tone shows no difference in the two groups, which suggested that the activity of IP3Rs-operated calcium channels is increased. These findings suggest that prenatal HS induces vascular dysfunction of thoracic arteries in fetal offspring by enhancing AT1R, LTCCs function and IP3Rs-associated calcium channels, providing new information regarding the impact of prenatal HS on the functional development of fetal vascular systems.

19734 L-type calcium channels in cerebellar neuron development and motor learning

ABSTRACT IMPACT: We aim to understand how LTCCs impact cerebellar function. OBJECTIVES/GOALS: L-type calcium channels (LTCCs) are important in activity-dependent neurite outgrowth, which comprises neurite initiation and elongation. We used cerebellar granule neurons (CGNs) to differentiate between LTCC effects on neurite initiation vs elongation. We also tested cerebellar function in mice lacking specific LTCCs with behavioral assays. METHODS/STUDY POPULATION: CGNs were cultured from 129SvEv mouse pups at P4-P6. Potassum chloride (50mM) was used to stimulate neuronal cultures for 24 hours. Isradipine (20nM) was added to culture medium to inhibit all LTCCs for 1 hour. For Cav1.2 deletion, we crossed Cav1.2 conditional knockout mice (Cav1.2-cKO) to Syn-Cre mice (for deletion in most neurons) or Atoh1-Cre mice (for deletion in CGNs). The Cav1.2-cKO line was maintained on a 129SvEv background. For constitutive Cav1.3 deletion, mice were maintained on a C57BL/6NTac. Behavioral tasks included open field, rotarod, and Erasmus Ladder. Data were analyzed with sexes combined and separated to assess for sex as a biological variable. Studies were analyzed by one-way ANOVA, two-way ANOVA, or generalized linear mixed model, where appropriate. RESULTS/ANTICIPATED RESULTS: CGNs exhibited an increase in neurite initiation but not elongation when stimulated with potassium chloride, consistent with previous reports of activity-dependent neurite outgrowth in this cell type. LTCC inhibition with isradipine blunted KCl-induced neurite initiation. We observed no change in the length of either primary or secondary neurites with isradipine treatment with or without KCl stimulation. In our behavioral experiments, we observed no deficits in open field, rotarod, or Erasmus Ladder when Cav1.2 was deleted in most neurons (driven by Syn-Cre expression) or in cerebellar granule neurons (driven by Atoh1-Cre expression). In contrast, loss of Cav1.3 was associated with impaired motor learning in the rotarod task without evidence of ataxia on Erasmus Ladder. DISCUSSION/SIGNIFICANCE OF FINDINGS: We show a specific role for LTCCs in activity-dependent CGN neurite initiation. While loss of Cav1.2 does not affect motor learning, loss of Cav1.3 does impair motor learning. Our results help expand our understanding of LTCC function in cerebellar neurodevelopment and function.

Rad-GTPase contributes to heart rate via L-type calcium channel regulation

Sinoatrial node cardiomyocytes (SANcm) possess automatic, rhythmic electrical activity. SAN rate is influenced by autonomic nervous system input, including sympathetic nerve increases of heart rate (HR) via activation of β-adrenergic receptor signaling cascade (β-AR). L-type calcium channel (LTCC) activity contributes to membrane depolarization and is a central target of β-AR signaling. Recent studies revealed that the small G-protein Rad plays a central role in β-adrenergic receptor directed modulation of LTCC. These studies have identified a conserved mechanism in which β-AR stimulation results in PKA-dependent Rad phosphorylation: depletion of Rad from the LTCC complex, which is proposed to relieve the constitutive inhibition of CaV1.2 imposed by Rad association. Here, using a transgenic mouse model permitting conditional cardiomyocyte selective Rad ablation, we examine the contribution of Rad to the control of SANcm LTCC current (ICa,L) and sinus rhythm. Single cell analysis from a recent published database indicates that Rad is expressed in SANcm, and we show that SANcm ICa,L was significantly increased in dispersed SANcm following Rad silencing compared to those from CTRL hearts. Moreover, cRadKO SANcm ICa,L was not further increased with β-AR agonists. We also evaluated heart rhythm in vivo using radiotelemetered ECG recordings in ambulating mice. In vivo, intrinsic HR is significantly elevated in cRadKO. During the sleep phase cRadKO also show elevated HR, and during the active phase there is no significant difference. Rad-deletion had no significant effect on heart rate variability. These results are consistent with Rad governing LTCC function under relatively low sympathetic drive conditions to contribute to slower HR during the diurnal sleep phase HR. In the absence of Rad, the tonic modulated SANcm ICa,L promotes elevated sinus HR. Future novel therapeutics for bradycardia targeting Rad – LTCC can thus elevate HR while retaining βAR responsiveness.

Calenduloside E suppresses calcium overload by promoting the interaction between L-type calcium channels and Bcl2-associated athanogene 3 to alleviate myocardial ischemia/reperfusion injury

IntroductionIntracellular calcium overload is an important contributor to myocardial ischemia/reperfusion (MI/R) injury. Total saponins of the traditional Chinese medicinal plant Aralia elata (Miq.) Seem. (AS) are beneficial for treating MI/R injury, and Calenduloside E (CE) is the main active ingredient of AS. ObjectivesThis study aimed to investigate the effects of CE on MI/R injury and determine its specific regulatory mechanisms. MethodsTo verify whether CE mediated cardiac protection in vivo and in vitro, we performed MI/R surgery in SD rats and subjected neonatal rat ventricular myocytes (NRVMs) to hypoxia-reoxygenation (HR). CE’s cardioprotective against MI/R injury was detected by Evans blue/TTC staining, echocardiography, HE staining, myocardial enzyme levels. Impedance and field potential recording, and patch-clamp techniques of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to detect the function of L-type calcium channels (LTCC). The mechanisms underlying between CE and LTCC was studied through western blot, immunofluorescence, and immunohistochemistry. Drug affinity responsive target stability (DARTS) and co-immunoprecipitation (co-IP) used to further clarify the effect of CE on LTCC and BAG3. ResultsWe found that CE protected against MI/R injury by inhibiting calcium overload. Furthermore, CE improved contraction and field potential signals of hiPSC-CMs and restored sarcomere contraction and calcium transient of adult rat ventricular myocytes (ARVMs). Moreover, patch-clamp data showed that CE suppressed increased L-type calcium current (ICa,L) caused by LTCC agonist, proving that CE could regulate calcium homeostasis through LTCC. Importantly, we found that CE promoted the interaction between LTCC and Bcl2-associated athanogene 3 (BAG3) by co-IP and DARTS. ConclusionOur results demonstrate that CE enhanced LTCC-BAG3 interaction to reduce MI/R induced-calcium overload, exerting a cardioprotective effect.

Nanoscale regulation of L-type calcium channels differentiates between ischemic and dilated cardiomyopathies.

BackgroundSubcellular localization and function of L-type calcium channels (LTCCs) play an important role in regulating contraction of cardiomyocytes. Understanding how this is affected by the disruption of transverse tubules during heart failure could lead to new insights into the disease.MethodsCardiomyocytes were isolated from healthy donor hearts, as well as from patients with cardiomyopathies and with left ventricular assist devices. Scanning ion conductance and confocal microscopy was used to study membrane structures in the cells. Super-resolution scanning patch-clamp was used to examine LTCC function in different microdomains. Computational modeling predicted the impact of these changes to arrhythmogenesis at the whole-heart level.FindingsWe showed that loss of structural organization in failing myocytes leads to re-distribution of functional LTCCs from the T-tubules to the sarcolemma. In ischemic cardiomyopathy, the increased LTCC open probability in the T-tubules depends on the phosphorylation by protein kinase A, whereas in dilated cardiomyopathy, the increased LTCC opening probability in the sarcolemma results from enhanced phosphorylation by calcium-calmodulin kinase II. LVAD implantation corrected LTCCs pathophysiological activity, although it did not improve their distribution. Using computational modeling in a 3D anatomically-realistic human ventricular model, we showed how LTCC location and activity can trigger heart rhythm disorders of different severity.InterpretationOur findings demonstrate that LTCC redistribution and function differentiate between disease aetiologies. The subcellular changes observed in specific microdomains could be the consequence of the action of distinct protein kinases.FundingThis work was supported by 10.13039/100000002NIH grant (ROI-HL 126802 to NT-JG) and British Heart Foundation (grant RG/17/13/33173 to JG, project grant PG/16/17/32069 to RAC). Funders had no role in study design, data collection, data analysis, interpretation, writing of the report

Metformin Shortens Prolonged QT Interval in Diabetic Mice by Inhibiting L-Type Calcium Current: A Possible Therapeutic Approach.

The incidence and mortality of cardiovascular disease in diabetic patients are 2-3 times higher than those in non-diabetic patients. Abnormal function of the L-type calcium channel in myocardial tissue might result in multiple cardiac disorders such as a prolonged QT interval. Therefore, QT prolongation is an independent risk factor of cardiovascular disease in patients with diabetes mellitus. Metformin, a hypoglycemic agent, is widely known to effectively reduce the occurrence of macrovascular diseases. The aim of the present study was to evaluate the effect of metformin on prolonged QT interval and to explore potential ionic mechanisms induced by diabetes. Diabetic mouse models were established with streptozotocin and an electrocardiogram was used to monitor the QT interval after 4 weeks of metformin treatment in each group. Action potential duration (APD) and L-type calcium current (ICa-L) were detected by patch-clamp in isolated mice ventricular cardiomyocytes and neonatal cardiomyocytes of mice. The expression levels of CACNA1C mRNA and Cav1.2 were measured by real-time PCR, western blot and immunofluorescence. A shortened QT interval was observed after 4 weeks of metformin treatment in diabetic mice. Patch-clamp results revealed that both APD and ICa-L were shortened in mouse cardiomyocytes. Furthermore, the expression levels of CACNA1C mRNA and Cav1.2 were decreased in the metformin group. The same results were also obtained in cultured neonatal mice cardiomyocytes. Overall, these results verify that metformin could shorten a prolonged QT interval by inhibiting the calcium current, suggesting that metformin may play a role in the electrophysiology underlying diabetic cardiopathy.

Junctophilin-2 tethers T-tubules and recruits functional L-type calcium channels to lipid rafts in adult cardiomyocytes.

AimIn cardiomyocytes, transverse tubules (T-tubules) associate with the sarcoplasmic reticulum (SR), forming junctional membrane complexes (JMCs) where L-type calcium channels (LTCCs) are juxtaposed to Ryanodine receptors (RyR). Junctophilin-2 (JPH2) supports the assembly of JMCs by tethering T-tubules to the SR membrane. T-tubule remodelling in cardiac diseases is associated with downregulation of JPH2 expression suggesting that JPH2 plays a crucial role in T-tubule stability. Furthermore, increasing evidence indicate that JPH2 might additionally act as a modulator of calcium signalling by directly regulating RyR and LTCCs. This study aimed at determining whether JPH2 overexpression restores normal T-tubule structure and LTCC function in cultured cardiomyocytes.Methods and resultsRat ventricular myocytes kept in culture for 4 days showed extensive T-tubule remodelling with impaired JPH2 localization and relocation of the scaffolding protein Caveolin3 (Cav3) from the T-tubules to the outer membrane. Overexpression of JPH2 restored T-tubule structure and Cav3 relocation. Depletion of membrane cholesterol by chronic treatment with methyl-β-cyclodextrin (MβCD) countered the stabilizing effect of JPH2 overexpression on T-tubules and Cav3. Super-resolution scanning patch-clamp showed that JPH2 overexpression greatly increased the number of functional LTCCs at the plasma membrane. Treatment with MβCD reduced LTCC open probability and activity. Proximity ligation assays showed that MβCD did not affect JPH2 interaction with RyR and the pore-forming LTCC subunit Cav1.2, but strongly impaired JPH2 association with Cav3 and the accessory LTCC subunit Cavβ2.ConclusionsJPH2 promotes T-tubule structural stability and recruits functional LTCCs to the membrane, most likely by directly binding to the channel. Cholesterol is involved in the binding of JPH2 to T-tubules as well as in the modulation of LTCC activity. We propose a model where cholesterol and Cav3 support the assembly of lipid rafts which provide an anchor for JPH2 to form JMCs and a platform for signalling complexes to regulate LTCC activity.

LITAF (Lipopolysaccharide-Induced Tumor Necrosis Factor) Regulates Cardiac L-Type Calcium Channels by Modulating NEDD (Neural Precursor Cell Expressed Developmentally Downregulated Protein) 4-1 Ubiquitin Ligase.

The turnover of cardiac ion channels underlying action potential duration is regulated by ubiquitination. Genome-wide association studies of QT interval identified several single-nucleotide polymorphisms located in or near genes involved in protein ubiquitination. A genetic variant upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor) gene prompted us to determine its role in modulating cardiac excitation. Optical mapping was performed in zebrafish hearts to determine Ca2+ transients. Live-cell confocal calcium imaging was performed on adult rabbit cardiomyocytes to determine intracellular Ca2+handling. L-type calcium channel (LTCC) current (ICa,L) was measured using whole-cell recording. To study the effect of LITAF on Cav1.2 (L-type voltage-gated calcium channel 1.2) channel expression, surface biotinylation, and Westerns were performed. LITAF interactions were studied using coimmunoprecipitation and in situ proximity ligation assay. LITAF knockdown in zebrafish resulted in a robust increase in calcium transients. Overexpressed LITAF in 3-week-old rabbit cardiomyocytes resulted in a decrease in ICa,L and Cavα1c abundance, whereas LITAF knockdown increased ICa,L and Cavα1c protein. LITAF-overexpressing decreases calcium transients in adult rabbit cardiomyocytes, which was associated with lower Cavα1c levels. In tsA201 cells, overexpressed LITAF downregulated total and surface pools of Cavα1c via increased Cavα1c ubiquitination and its subsequent lysosomal degradation. We observed colocalization between LITAF and LTCC in tsA201 and cardiomyocytes. In tsA201, NEDD (neural precursor cell expressed developmentally downregulated protein) 4-1, but not its catalytically inactive form NEDD4-1-C867A, increased Cavα1c ubiquitination. Cavα1c ubiquitination was further increased by coexpressed LITAF and NEDD4-1 but not NEDD4-1-C867A. NEDD4-1 knockdown abolished the negative effect of LITAF on ICa,L and Cavα1c levels in 3-week-old rabbit cardiomyocytes. Computer simulations demonstrated that a decrease of ICa,L current associated with LITAF overexpression simultaneously shortened action potential duration and decreased calcium transients in rabbit cardiomyocytes. LITAF acts as an adaptor protein promoting NEDD4-1-mediated ubiquitination and subsequent degradation of LTCC, thereby controlling LTCC membrane levels and function and thus cardiac excitation.

Abstract 807: Rad Ablation as a Treatment to Target Cardiac Inotropy via L-type Calcium Channel Function

The L-type Ca 2+ channel current (I Ca,L ) provides trigger Ca 2+ to contribute to cardiac contraction. Rad GTPase associates with the L-type Ca 2+ channel (LTCC) and serves as an endogenous inhibitor of LTCC activity. Overexpression of Rad blocks I Ca,L ; absence of Rad increases I Ca,L . Rad attenuates β-adrenergic receptor (β-AR) signaling. Chronic β-AR stimulation associates with Ca 2+ mishandling and can promote signaling that progresses towards heart failure. Early studies of global, constitutive Rad-knockout mice (gRadKO) suggested that elevated Ca 2+ dynamics leads to pathological cardiac hypertrophy; however, Rad is also expressed in non-cardiac tissues. Our objective is to test the hypothesis that increased myocardial I Ca,L via Rad deletion safely enhances cardiac function without driving pathological remodeling. We created a cardiac-restricted inducible Rad knockout mouse (Rad Δ/Δ ). In vivo function was measured with echocardiography. We examined I Ca,L through whole cell configuration of the patch clamp technique, assessed Ca 2+ handling, and sarcomere dynamics. Unlike gRadKO, Rad Δ/Δ showed no elevation of fetal gene program, nor fibrosis, and no change to aortic pressure. Rad Δ/Δ had a sustained increase of inotropy without structural or functional remodeling (EF: Rad Δ/Δ =76 ± 2%, n=16; Rad fl/fl =59 ± 4%, n=7; p=0.001.) I Ca,L was significantly increased, with Rad loss mirroring a β-AR modulated phenotype on basal I Ca,L (max. conductance: Rad Δ/Δ =254 ± 19 pS/pF, n=15; Rad fl/fl =144 ± 12 pS/pF, n=18; p<10 -4 ). Contrary to models of chronic β-AR stimulation, Rad Δ/Δ retained β-AR signaling shown in vivo using isoproterenol, and by preserved phosphorylation of protein regulators of Ca 2+ reuptake and contractility. Rad Δ/Δ cardiomyocytes show enhanced cytosolic Ca 2+ handling (Decay of Ca 2+ transient: Rad Δ/Δ = 0.07 ± 0.003 (F 340 /F 380 )/s, n=67, Rad fl/fl = 0.10 ± 0.005, n=69; p<10 -4 ), increased contractile function, and elevated SERCA2a expression. These new findings challenge the canonical assumption that increased myocardial Ca 2+ necessarily promotes pathology. We conclude that cardiac hypertrophy in gRadKO was caused by non-cardiac tissue effects, and myocardial Rad deletion is a promising cardiac inotropic therapeutic direction.

AKAP79/150 recruits the transcription factor NFAT to regulate signaling to the nucleus by neuronal L-type Ca2+ channels.

In neurons, regulation of activity-dependent transcription by the nuclear factor of activated T-cells (NFAT) depends upon Ca2+ influx through voltage-gated L-type calcium channels (LTCC) and NFAT translocation to the nucleus following its dephosphorylation by the Ca2+-dependent phosphatase calcineurin (CaN). CaN is recruited to the channel by A-kinase anchoring protein (AKAP) 79/150, which binds to the LTCC C-terminus via a modified leucine-zipper (LZ) interaction. Here we sought to gain new insights into how LTCCs and signaling to NFAT are regulated by this LZ interaction. RNA interference–mediated knockdown of endogenous AKAP150 and replacement with human AKAP79 lacking its C-terminal LZ domain resulted in loss of depolarization-stimulated NFAT signaling in rat hippocampal neurons. However, the LZ mutation had little impact on the AKAP–LTCC interaction or LTCC function, as measured by Förster resonance energy transfer, Ca2+ imaging, and electrophysiological recordings. AKAP79 and NFAT coimmunoprecipitated when coexpressed in heterologous cells, and the LZ mutation disrupted this association. Critically, measurements of NFAT mobility in neurons employing fluorescence recovery after photobleaching and fluorescence correlation spectroscopy provided further evidence for an AKAP79 LZ interaction with NFAT. These findings suggest that the AKAP79/150 LZ motif functions to recruit NFAT to the LTCC signaling complex to promote its activation by AKAP-anchored calcineurin.

Degradation of T-Tubular Microdomains and Altered cAMP Compartmentation Lead to Emergence of Arrhythmogenic Triggers in Heart Failure Myocytes: An in silico Study.

Heart failure (HF) is one of the most common causes of morbidity and mortality worldwide. Although many patients suffering from HF die from sudden cardiac death caused by arrhythmias, the mechanism linking HF remodeling to an increased arrhythmogenic propensity remains incomplete. HF is typically characterized by a progressive loss of transverse tubule (T-tubule) domains, which leads to an altered distribution of L-type calcium channels (LTCCs). Microdomain degradation also causes the disruption of the β2 adrenergic receptor (β2AR) and phosphodiesterase (PDE) signaling localization, normally confined to the dyadic space. The goal of this study was to analyze how these subcellular changes affect the function of LTCCs and lead to the emergence of ventricular cell-level triggers of arrhythmias. To accomplish this, we developed a novel computational model of a human ventricular HF myocyte in which LTCCs were divided into six different populations, based on their location and signaling environment they experience. To do so, we included T-tubular microdomain remodeling which led to a subset of LTCCs to be redistributed from the T-tubular to the surface membrane and allowed for different levels of phosphorylation of LTCCs by PKA, based on the presence of β2ARs and PDEs. The model was used to study the behavior of the LTCC current (ICaL) under basal and sympathetic stimulation and its effect on cellular action potential. Our results showed that channels redistributed from the T-tubular membrane to the bulk of the sarcolemma displayed an altered function in their new, non-native signaling domain. Incomplete calcium dependent inactivation, which resulted in a longer-lasting and larger-in-magnitude LTCC current, was observed when we decoupled LTCCs from ryanodine receptors and removed them from the dyadic space. The magnitude of the LTCC current, especially in the surface sarcolemma, was also increased via phosphorylation by the redistributed β2ARs and PDEs. These changes in LTCC current led to the development of early afterdepolarizations. Thus, our study shows that altered LTCC function is a potential cause for the emergence of cell-level triggers of arrhythmia, and that β2ARs and PDEs present useful therapeutic targets for treatment of HF and prevention of sudden cardiac death.

Subplasmalemmal hydrogen peroxide triggers calcium influx in gonadotropes

Gonadotropin-releasing hormone (GnRH) stimulation of its eponymous receptor on the surface of endocrine anterior pituitary gonadotrope cells (gonadotropes) initiates multiple signaling cascades that culminate in the secretion of luteinizing and follicle-stimulating hormones, which have critical roles in fertility and reproduction. Enhanced luteinizing hormone biosynthesis, a necessary event for ovulation, requires a signaling pathway characterized by calcium influx through L-type calcium channels and subsequent activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK). We previously reported that highly localized subplasmalemmal calcium microdomains produced by L-type calcium channels (calcium sparklets) play an essential part in GnRH-dependent ERK activation. Similar to calcium, reactive oxygen species (ROS) are ubiquitous intracellular signaling molecules whose subcellular localization determines their specificity. To investigate the potential influence of oxidant signaling in gonadotropes, here we examined the impact of ROS generation on L-type calcium channel function. Total internal reflection fluorescence (TIRF) microscopy revealed that GnRH induces spatially restricted sites of ROS generation in gonadotrope-derived αT3-1 cells. Furthermore, GnRH-dependent stimulation of L-type calcium channels required intracellular hydrogen peroxide signaling in these cells and in primary mouse gonadotropes. NADPH oxidase and mitochondrial ROS generation were each necessary for GnRH-mediated stimulation of L-type calcium channels. Congruently, GnRH increased oxidation within subplasmalemmal mitochondria, and L-type calcium channel activity correlated strongly with the presence of adjacent mitochondria. Collectively, our results provide compelling evidence that NADPH oxidase activity and mitochondria-derived hydrogen peroxide signaling play a fundamental role in GnRH-dependent stimulation of L-type calcium channels in anterior pituitary gonadotropes.

Physical interaction of junctophilin and the CaV1.1 C terminus is crucial for skeletal muscle contraction

Close physical association of CaV1.1 L-type calcium channels (LTCCs) at the sarcolemmal junctional membrane (JM) with ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR) is crucial for excitation-contraction coupling (ECC) in skeletal muscle. However, the molecular mechanism underlying the JM targeting of LTCCs is unexplored. Junctophilin 1 (JP1) and JP2 stabilize the JM by bridging the sarcolemmal and SR membranes. Here, we examined the roles of JPs in localization and function of LTCCs. Knockdown of JP1 or JP2 in cultured myotubes inhibited LTCC clustering at the JM and suppressed evoked Ca2+ transients without disrupting JM structure. Coimmunoprecipitation and GST pull-down assays demonstrated that JPs physically interacted with 12-aa residues in the proximal C terminus of the CaV1.1. A JP1 mutant lacking the C terminus including the transmembrane domain (JP1ΔCT) interacted with the sarcolemmal/T-tubule membrane but not the SR membrane. Expression of this mutant in adult mouse muscles in vivo exerted a dominant-negative effect on endogenous JPs, impairing LTCC-RyR coupling at triads without disrupting JM morphology, and substantially reducing Ca2+ transients without affecting SR Ca2+ content. Moreover, the contractile force of the JP1ΔCT-expressed muscle was dramatically reduced compared with the control. Taken together, JPs recruit LTCCs to the JM through physical interaction and ensure robust ECC at triads in skeletal muscle.