Calcium Channels In Cardiomyocytes Research Articles

Pharmacological Blockade of High Threshold L-Type Calcium Channels in Cardiomyocytes Restores Respiratory Chain Activity Impaired by Vibration

Pharmacological Blockade of High Threshold L-Type Calcium Channels in Cardiomyocytes Restores Respiratory Chain Activity Impaired by Vibration

Analysis of heroin effects on calcium channels in rat cardiomyocytes based on transcriptomics and metabolomics.

Heroin can cause damage to many human organs, possibly leading to different types of arrhythmias and abnormal electrophysiological function of the heart muscle and the steady state of calcium-ion channels. We explored cardiomyocytes treated with heroin and the effect on calcium-ion channels. Transcriptomics and metabolomics were used to screen for differential genes and metabolite alterations after heroin administration to jointly analyze the effect of heroin on calcium channels in cardiomyocytes. Cardiomyocytes from primary neonatal rats were cultured in vitro and were treated with different concentrations of heroin to observe the changes in morphology and spontaneous beat frequency and rhythm by a patch clamp technique. Transcriptomic studies selected a total of 1,432 differentially expressed genes, 941 upregulated and 491 downregulated genes in rat cardiomyocytes from the control and drug intervention groups. Gene Ontology functional enrichment showed that 1,432 differential genes selected by the two groups were mainly involved in the regulation of the multicellular organismal process, response to external stimulus, myofibril, inflammatory response, muscle system process, cardiac muscle contraction, etc. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that these genes were mainly concentrated in cardiac muscle contraction, osteoclast differentiation, adrenergic signaling in cardiomyocytes, dilated cardiomyopathy, hypertrophic cardiomyopathy, and other important pathways. Metabolomic testing further suggested that cardiomyocyte metabolism was severely affected after heroin intervention. After the treatment with heroin, the L-type calcium channel current I-V curve was up-shifted, the peak value was significantly lower than that of the control group, action potential duration 90 was significantly increased in the action potential, resting potential negative value was lowered, and action potential amplitude was significantly decreased in cardiomyocytes. In this study, heroin could cause morphological changes in primary cardiomyocytes of neonatal rats and electrophysiological function. Heroin can cause myocardial contraction and calcium channel abnormalities, damage the myocardium, and change the action potential and L-type calcium channel.

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter.

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs). These iCMs, when generated in situ in an infarcted heart, integrate electrically and mechanically with the surrounding myocardium, leading to a reduction in scar size and an improvement in heart function. Because of the relatively low reprogramming efficiency, purity, and quality of the iCMs, characterization of iCMs remains a challenge. The currently used methods in this field, including flow cytometry, immunocytochemistry, and qPCR, mainly focus on cardiac-specific gene and protein expression but not on the functional maturation of iCMs. Triggered by action potentials, the opening of voltage-gated calcium channels in cardiomyocytes leads to a rapid influx of calcium into the cell. Therefore, quantifying the rate of calcium influx is a promising method to evaluate cardiomyocyte function. Here, the protocol introduces a method to evaluate iCM function by calcium (Ca2+) flux. An αMHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain was established by crossing Tg(Myh6-cre)1Jmk/J (referred to as Myh6-Cre below) with Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as Rosa26A-Flox-Stop-Flox-GCaMP3 below) mice. Neonatal cardiac fibroblasts (NCFs) from P0-P2 neonatal mice were isolated and cultured in vitro, and a polycistronic construction of MGT was introduced to NCFs, which led to their reprogramming to iCMs. Because only successfully reprogrammed iCMs will express GCaMP3 reporter, the functional maturation of iCMs can be visually assessed by Ca2+ flux with fluorescence microscopy. Compared with un-reprogrammed NCFs, NCF-iCMs showed significant calcium transient flux and spontaneous contraction, similar to CMs. This protocol describes in detail the mouse strain establishment, isolation and selection of neonatal mice hearts, NCF isolation, production of retrovirus for cardiac reprogramming, iCM induction, the evaluation of iCM Ca2+ flux using our reporter line, and related statistical analysis and data presentation. It is expected that the methods described here will provide a valuable platform to assess the functional maturation of iCMs for cardiac reprogramming studies.

Luteolin improves heart preservation through inhibiting hypoxia-dependent L-type calcium channels in cardiomyocytes.

The current study aimed to evaluate whether luteolin could improve long-term heart preservation; this was achieved by evaluating the heart following long-term storage in University of Wisconsin solution (the control group) and in solutions containing three luteolin concentrations. The effects of different preservation methods were evaluated with respect to cardiac function while hearts were in custom-made ex vivo Langendorff perfusion systems. Different preservation methods were evaluated with respect to the histology, ultrastructure and apoptosis rate of the hearts, and the function of cardiomyocytes. In the presence of luteolin, the rate pressure product of the left ventricle was increased within 60 min of reperfusion following a 12-h preservation, coronary flow was higher within 30 min of reperfusion, cardiac contractile function was higher throughout reperfusion following 12- and 18-h preservations, and the left ventricle peak systolic pressure was significantly higher compared with the control group (all P<0.05). The expression levels of apoptosis regulator Bax and apoptosis regulator Bcl-2 in the luteolin groups were significantly decreased and increased, respectively. Lactate dehydrogenase, creatine kinase and malondialdehyde enzymatic activity was increased following long-term storage, while the activity of superoxide dismutase was significantly decreased. Furthermore, luteolin inhibited L-type calcium currents in ventricular myocytes under hypoxia conditions. Thus, luteolin demonstrated protective effects during long-term heart preservation in what appeared to be a dose-dependent manner, which may be accomplished through inhibiting hypoxia-dependent L-type calcium channels.

Wnt Signaling Inhibits Calcium Channels in Cardiomyocytes

Wnt Signaling Inhibits Calcium Channels in Cardiomyocytes

Meet the First Authors.

Meet the First Authors.

Spatial and Functional Interactions between SK Channels and L-Type Calcium Channels in Cardiomyocytes

Small conductance Ca2+-activated K+ (SK) channels are gated solely by intracellular Ca2+ and hence, provide a critical link between changes in intracellular Ca2+ and membrane potentials. The identification and functional expression of SK channels in the heart provided additional insights into the regulation of cardiac excitability. Indeed, the crosstalk between Ca2+ signaling and SK channels is critical for shaping cardiac repolarization. Our prior studies documented the physical coupling between SK channels and L-type Ca2+ channels (LTCCs). In addition, functional dependence of SK channel on sarcoplasmic reticulum has also been demonstrated. However, the spatial and functional interactions are less well understood. We hypothesize that LTCCs provide the immediate Ca2+ domain for the activation of cardiac SK channels. Using stimulated emission depletion (STED) microscopy, we gained super-resolution images of SK channels, LTCCs and ryanodine receptors (RyR2) in rabbit ventricular myocytes, revealing the spatial coupling of the three molecules. Moreover, we designed a voltage-clamp protocol to test the functional coupling between SK and LTCCs. EGTA or BAPTA were applied to identify the intracellular Ca2+ domains for SK channel activation. SK currents were activated by Ca2+ influx via LTCCs after depletion of sarcoplasmic reticulum (SR) Ca2+ store. BAPTA removed the functional coupling between SKs and LTCCs. In addition, alterations in SK channel activation kinetics were observed after SR Ca2+ depletion. We conclude that the spatial proximity among SK, LTCCs and RyR2 provides the structural basis for the functional complex formation for SK channel activation. Ca2+ influx through LTCCs provides Ca2+ microdomain for SK channel activation as well as a joint contribution from LTCCs and SR for SK channel activation in cardiomyocytes.Funded by NIH (R01 HL085727 and R01 HL085844 to NC), VA (I01 BX000576 to NC) and AHA (14BGIA18870087 to XZ).

Abstract 16089: Functional Coupling of SK Channels and L-type Calcium Channels in Cardiomyocytes

Background. Small conductance Ca 2+ -activated K + (SK) channels play significant roles in regulating the excitability of cardiomyocytes (CMs). SK channels are unique in that they are gated solely by intracellular Ca 2+ and hence, function to integrate intracellular Ca 2+ and membrane potentials on a beat-to-beat basis in the heart. Our previous studies revealed that cardiac SK2 channels coupled with L-type Ca 2+ channels (LTCCs) through a physical bridge, α-actinin2, suggesting that LTCCs may be functionally coupled with SK2 channels by providing local Ca 2+ domain to activate the SK channels. However, a recent study suggested that sarcoplasmic reticulum (SR) Ca 2+ release is necessary and sufficient for the activation of cardiac SK channels. The objective of the study is to examine the mechanisms of SK channel activation in native CMs. Methods and Results. By using a voltage-clamp protocol in rabbit CMs to activate LTCCs followed immediately by a test voltage to monitor the SK currents, we recorded apamin-sensitive SK currents in response to the Ca 2+ influx. By altering the duration of activation of LTCCs, the activation of SK currents as a function of the increasing Ca 2+ influx was quantified. EGTA or BAPTA were then used to test the intracellular Ca 2+ domain necessary for SK channel activation. BAPTA but not EGTA completely disengaged the coupling between LTCCs and SK channels. SK currents coupled with Ca 2+ influx via LTCCs continued to be elicited after application of caffeine, ryanodine or thapsigargin to deplete SR Ca 2+ store, suggesting that LTCCs provide the immediate Ca 2+ microdomain for the activation of SK channels in CMs. Super-resolution imaging of SK2, Ca v 1.2, and ryanodine receptor 2 (RyR2) using stimulated emission depletion (STED) microscopy was performed to directly quantify the distance among SK, LTCC and RyR2 in CMs. Conclusion. Ca 2+ influx through LTCCs provides the immediate and efficient Ca 2+ microdomain for the activation of SK channels. Furthermore, changes in SK channel activation kinetics following SR Ca 2+ depletion suggest both LTCC-mediated Ca 2+ influx and SR Ca 2+ release are necessary for the activation of cardiac SK channels. Funded by NIH (R01 HL085727 and R01 HL085844 to NC), VA (I01 BX000576 to NC) and AHA (14BGIA18870087 to XZ).

Vasorelaxation induced by dodoneine is mediated by calcium channels blockade and carbonic anhydrase inhibition on vascular smooth muscle cells

Ethnopharmacological relevanceDodoneine (Ddn) is one of the active compounds identified from Agelanthus dodoneifolius (DC.) Polhill and Wiens, a medicinal plant used in traditional medicine for the treatment of hypertension. This dihydropyranone exerts hypotensive and vasorelaxant effects on rats, and two molecular targets have been characterized: the carbonic anhydrase and the L-type calcium channel in cardiomyocytes with biochemical and electrophysiological techniques, respectively. To further evaluate the involvement of these two molecular targets in vasorelaxation, the effect of Ddn on rat vascular smooth muscle was investigated. Material and methodsThe effects of Ddn on L-type calcium current and on resting membrane potential were characterized in A7r5 cell line using the whole-cell patch-clamp configuration. The molecular identities of carbonic anhydrase isozymes in smooth muscle cells were examined with RT-PCR. Vascular response was measured on rat aortic rings in an organ bath apparatus and the effect of Ddn on intracellular pH was determined by flow cytometry using the pH-sensitive fluorescent probe BCECF-AM [2,7-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester]. Results100µM Ddn reduced calcium current density of about 30%. In addition, carbonic anhydrase II, III, XIII and XIV were shown to be expressed in rat aorta and inhibited in smooth muscle cells by Ddn. This inhibition resulted in a rise in pHi of about 0.31, leading to KCa channel activation, thereby inducing membrane hyperpolarization and vasorelaxation. The results of vascular reactivity experiments obtained with pharmacological tools acting on the L-type calcium current and carbonic anhydrase suggest that Ddn produces its vasorelaxant effect via the inhibition of these two molecular targets. ConclusionThis study demonstrates that Ddn induced vasorelaxation by targeting two proteins involved in the modulation of excitation-contraction coupling: L-type calcium channels and carbonic anhydrase.

Silencing of striatin alters the association of caveolin‐3 with the L‐type calcium channel in cardiomyocytes.

Striatin (STRN) is a dynamic protein containing binding domains for caveolin (Cav), protein phosphatases 2A (PP2A), and calmodulin (CaM). Although these proteins regulate the kinetics of the L‐type calcium channel (LCC), the role of STRN in this context remains unknown. We have previously reported that STRN can modulate the intracellular calcium and the contraction rate of cultured cardiomyocytes (CMs). Here we show that STRN is highly expressed in adult rat ventricles as compared to atria. Biochemical analysis revealed that neither the knockdown of STRN (shRNA) nor its overexpression in CMs modulated the expression level of Cav‐3, PP2A, or LCC. Interestingly, silencing of STRN enhanced the association of Cav‐3 with CaM but not PP2A. In contrast, the overexpression of STRN significantly reduced the interaction of Cav‐3 with CaM and PP2A. In both cases, the amount of LCC interacting with STRN/Cav‐3/CaM/PP2A complex was unchanged. Taken together, our data suggest that the regulation of intracellular calcium by STRN may be due to alterations in Caveolin‐3 binding to CaM/PP2A/LCC complex thus regulating the kinetics of LCC in cardiomyocytes.

Hydrogen Sulfide Targets the Cys320/Cys529 Motif in Kv4.2 to Inhibit the Ito Potassium Channels in Cardiomyocytes and Regularizes Fatal Arrhythmia in Myocardial Infarction.

The mechanisms underlying numerous biological roles of hydrogen sulfide (H2S) remain largely unknown. We have previously reported an inhibitory role of H2S in the L-type calcium channels in cardiomyocytes. This prompts us to examine the mechanisms underlying the potential regulation of H2S on the ion channels. H2S showed a novel inhibitory effect on Ito potassium channels, and this effect was blocked by mutation at the Cys320 and/or Cys529 residues of the Kv4.2 subunit. H2S broke the disulfide bridge between a pair of oxidized cysteine residues; however, it did not modify single cysteine residues. H2S extended action potential duration in epicardial myocytes and regularized fatal arrhythmia in a rat model of myocardial infarction. H2S treatment significantly increased survival by ∼1.4-fold in the critical 2-h time window after myocardial infarction with a protection against ventricular premature beats and fatal arrhythmia. However, H2S did not change the function of other ion channels, including IK1 and INa. H2S targets the Cys320/Cys529 motif in Kv4.2 to regulate the Ito potassium channels. H2S also shows a potent regularizing effect against fatal arrhythmia in a rat model of myocardial infarction. The study provides the first piece of evidence for the role of H2S in regulating Ito potassium channels and also the specific motif in an ion channel labile for H2S regulation.

Enhanced currents through L-type calcium channels in cardiomyocytes disturb the electrophysiology of the dystrophic heart

Duchenne muscular dystrophy (DMD), induced by mutations in the gene encoding for the cytoskeletal protein dystrophin, is an inherited disease characterized by progressive muscle weakness. Besides the relatively well characterized skeletal muscle degenerative processes, DMD is also associated with cardiac complications. These include cardiomyopathy development and cardiac arrhythmias. The current understanding of the pathomechanisms in the heart is very limited, but recent research indicates that dysfunctional ion channels in dystrophic cardiomyocytes play a role. The aim of the present study was to characterize abnormalities in L-type calcium channel function in adult dystrophic ventricular cardiomyocytes. By using the whole cell patch-clamp technique, the properties of currents through calcium channels in ventricular cardiomyocytes isolated from the hearts of normal and dystrophic adult mice were compared. Besides the commonly used dystrophin-deficient mdx mouse model for human DMD, we also used mdx-utr mice, which are both dystrophin- and utrophin-deficient. We found that calcium channel currents were significantly increased, and channel inactivation was reduced in dystrophic cardiomyocytes. Both effects enhance the calcium influx during an action potential (AP). Whereas the AP in dystrophic mouse cardiomyocytes was nearly normal, implementation of the enhanced dystrophic calcium conductance in a computer model of a human ventricular cardiomyocyte considerably prolonged the AP. Finally, the described dystrophic calcium channel abnormalities entailed alterations in the electrocardiograms of dystrophic mice. We conclude that gain of function in cardiac L-type calcium channels may disturb the electrophysiology of the dystrophic heart and thereby cause arrhythmias.

Manipulating L-type calcium channels in cardiomyocytes using split-intein protein transsplicing

Manipulating expression of large genes (>6 kb) in adult cardiomyocytes is challenging because these cells are only efficiently transduced by viral vectors with a 4-7 kb packaging capacity. This limitation impedes understanding structure-function mechanisms of important proteins in heart. L-type calcium channels (LTCCs) regulate diverse facets of cardiac physiology including excitation-contraction coupling, excitability, and gene expression. Many important questions about how LTCCs mediate such multidimensional signaling are best resolved by manipulating expression of the 6.6 kb pore-forming α1C-subunit in adult cardiomyocytes. Here, we use split-intein-mediated protein transsplicing to reconstitute LTCC α1C-subunit from two distinct halves, overcoming the difficulty of expressing full-length α1C in cardiomyocytes. Split-intein-tagged α1C fragments encoding dihydropyridine-resistant channels were incorporated into adenovirus and reconstituted in cardiomyocytes. Similar to endogenous LTCCs, recombinant channels targeted to dyads, triggered Ca(2+) transients, associated with caveolin-3, and supported β-adrenergic regulation of excitation-contraction coupling. This approach lowers a longstanding technical hurdle to manipulating large proteins in cardiomyocytes.

Correction: Gαi2- and Gαi3-Specific Regulation of Voltage-Dependent L-Type Calcium Channels in Cardiomyocytes

An author was omitted from the list. The ninth author should be Lutz Bjrnbaumer. The affiliation of this author is: Laboratory of Neurobiology, National Institutes of Health, 111 TW Alexander Drive, North Carolina, United States of America Additional Funding information for this author is: Research supported in part by the Intramural Research Program of the NIH, Project 201-ES-101643. The Author Contributions of this author were: contributed reagents/materials analysis tools.

Role of the scaffold protein striatin in regulating the excitation‐contraction coupling in cardiomyocytes

Striatin (STRN) is a multivalent protein with dynamic domains including caveolin (Cav) and calmodulin (CaM) binding sites; both of which are key regulators of the L‐type calcium channel (LCC). We have previously reported that the formation of the STRN/Cav/CaM complex was calcium sensitive and that overexpression of striatin resulted in a sustained elevation of KCl‐induced intracellular calcium increase. We aimed, in this study, to show whether STRN interacts with LCC and PP2A to regulate cardiomyocyte contractility. Herein we report that both protein phosphatase 2A (PP2A) and the LCC were associated with STRN/Cav/CaM complex and that the interaction between all these components was calcium sensitive (abolished by calcium chelator EGTA). In addition, we also show that overexpression of striatin (adenoviral delivery) in cultured cardiomyocytes increased the rate of contraction by ~ 3 fold when compared to control cells (non‐target adenovirus). Preliminary data showed that knockdown of striatin (shRNA targeting STRN) enhanced the interaction of CaM and Caveolin‐3 suggesting a central role for STRN in articulating the STRN/Cav/CaM/LCC/PP2A complex. Collectively, our data suggest that striatin may emerge as a novel regulator of the L‐type calcium channel in cardiomyocytes and consequently regulate their contractility.This work was supported by an operating grant from KFSHRC.

Effects of &lt;i&gt;Leonurine&lt;/i&gt; on L-Type Calcium Channel in Rat Ventricular Myocytes

Leonurine (Leo) is a special alkaloid principle of Herba leonuri that has recently been suggested to improve cardiovascular functions. To date, there is no direct ionic evidence of Leo on regulating calcium channels in the heart. In the present study, we examined the effects of Leo on action potentials and membrane currents recorded from isolated rat ventricular myocytes with the whole-cell patch clamp technique. Leo 100 µM shortened the action potential duration in a dose-dependent manner. Leo up to 200 µM had no significant effect on the Na+ current (INa) and K+ current (IK). However, Leo depressed the L-type Ca2+ current (ICa,L). In the presence of 20 and 100 µM Leo, the current density was decreased and the voltage at half maximal inactivation V0.5 shift to more negative potential. The recovery time constant was also delayed. In addition, the transcription and protein expression levels of L-type calcium channel (Cav1.2) in primary cultured neonatal myocytes from Sprague-Dawley rats were reduced by Leo treatment in a dose-dependent fashion as assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assays. We conclude that Leo inhibits L-type calcium channels in cardiomyocytes.

Gαi2- and Gαi3-Specific Regulation of Voltage-Dependent L-Type Calcium Channels in Cardiomyocytes

BackgroundTwo pertussis toxin sensitive Gi proteins, Gi2 and Gi3, are expressed in cardiomyocytes and upregulated in heart failure. It has been proposed that the highly homologous Gi isoforms are functionally distinct. To test for isoform-specific functions of Gi proteins, we examined their role in the regulation of cardiac L-type voltage-dependent calcium channels (L-VDCC).MethodsVentricular tissues and isolated myocytes were obtained from mice with targeted deletion of either Gαi2 (Gαi2 −/−) or Gαi3 (Gαi3 −/−). mRNA levels of Gαi/o isoforms and L-VDCC subunits were quantified by real-time PCR. Gαi and Cavα1 protein levels as well as protein kinase B/Akt and extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation levels were assessed by immunoblot analysis. L-VDCC function was assessed by whole-cell and single-channel current recordings.ResultsIn cardiac tissue from Gαi2 −/− mice, Gαi3 mRNA and protein expression was upregulated to 187±21% and 567±59%, respectively. In Gαi3 −/− mouse hearts, Gαi2 mRNA (127±5%) and protein (131±10%) levels were slightly enhanced. Interestingly, L-VDCC current density in cardiomyocytes from Gαi2 −/− mice was lowered (−7.9±0.6 pA/pF, n = 11, p<0.05) compared to wild-type cells (−10.7±0.5 pA/pF, n = 22), whereas it was increased in myocytes from Gαi3 −/− mice (−14.3±0.8 pA/pF, n = 14, p<0.05). Steady-state inactivation was shifted to negative potentials, and recovery kinetics slowed in the absence of Gαi2 (but not of Gαi3) and following treatment with pertussis toxin in Gαi3 −/−. The pore forming Cavα1 protein level was unchanged in all mouse models analyzed, similar to mRNA levels of Cavα1 and Cavβ2 subunits. Interestingly, at the cellular signalling level, phosphorylation assays revealed abolished carbachol-triggered activation of ERK1/2 in mice lacking Gαi2.ConclusionOur data provide novel evidence for an isoform-specific modulation of L-VDCC by Gαi proteins. In particular, loss of Gαi2 is reflected by alterations in channel kinetics and likely involves an impairment of the ERK1/2 signalling pathway.

Hydrogen sulphide is an inhibitor of L-type calcium channels and mechanical contraction in rat cardiomyocytes

Hydrogen sulphide (H(2)S) is an endogenously generated gaseous transmitter that has recently been suggested to regulate cardiovascular functions. To date, there is no direct evidence for a potential role of H(2)S in regulating calcium channels in the heart. The present study aims to examine the hypothesis that H(2)S is a novel inhibitor of the L-type calcium channel current (I(Ca,L)). Electrophysiological measurements were performed in cardiomyocytes isolated from Wistar-Kyoto and spontaneously hypertensive rats. Bath application of 100 microM NaHS (a H(2)S donor) significantly reduced the time required for the repolarization of the action potential. Inhibition of the peak I(Ca,L) by NaHS was determined to be concentration-dependent (25, 50, 100, 200, and 400 microM). NaHS inhibited the recovery from depolarization-induced inactivation. Electric field-induced [Ca(2+)]i transients and contraction of single cardiomyocytes and isolated papillary muscles were reduced by NaHS treatment. In contrast, caffeine induced an increase in [Ca(2+)]i that was not altered by NaHS. NaHS had no effect on the K(ATP) current or on the levels of cAMP and cGMP in the current study. H(2)S is a novel inhibitor of L-type calcium channels in cardiomyocytes. Moreover, H(2)S-induced inhibition of [Ca(2+)]i appears to be a secondary effect owing to its initial action towards I(Ca,L). The inhibitory effect of H(2)S on I(Ca,L) requires further investigation, particularly in the exploration of new pathways involved in cardiac calcium homeostasis and disease pathology.

NGP1-01 is a Brain-permeable Dual Blocker of Neuronal Voltage- and Ligand-operated Calcium Channels

Calcium overload of neurons leads to cell death and is a key feature in neurodegenerative diseases. The polycyclic amine NGP1-01 blocks L-type voltage operated calcium channels in cardiomyocytes. Here, we tested whether NGP1-01 blocks neuronal calcium channels. NGP1-01 (1 microM) inhibited depolarization-induced calcium influx by 78% in cortical neurons preloaded with fura-2 AM, with a potency similar to nimodipine. NGP1-01 (1 microM) also inhibited N-methyl-D: -aspartate (NMDA)-induced (1 mM) calcium influx by 52%, only slightly less potent than memantine. Using in vivo-microdialysis, we monitored choline release during NMDA infusion as a measure of excitotoxic membrane breakdown. Intraperitoneal injection of NGP1-01 (40 mg/kg) reduced NMDA-induced membrane breakdown by 31% (P < 0.01) while memantine (10 mg/kg) reduced choline release by 40%. Our results demonstrate that NGP1-01 simultaneously blocks both major neuronal calcium channels and is sufficiently brain-permeable. We conclude that NGP1-01 is a promising lead structure for a new class of dual-mechanism neuroprotective agents.

Single-channel gating and regulation of human L-type calcium channels in cardiomyocytes of transgenic mice

Overexpression of human cardiac L-type Ca 2+ channel pores (hCa v1.2) in mice causes heart failure. Earlier studies showed Ca v1.2-mRNA increase by 2.8-fold, but whole-cell current density enhancement by ⩽1.5-fold only. Three possible explanations were examined: (1) poor translation of hCa v1.2 and of its accessory subunits, (2) altered sarcolemmal insertion of functional channels, and (3) lower single-channel activity of overexpressed channels. Western blots revealed a 2.7-fold increase of Ca v1.2 protein in transgenic myocytes, but less enhanced expression of β 1a and β 1b subunits. β 2 and α 2/δ were significantly lowered. Density of functional channels was increased by 3.0-fold. Single-channel gating was impaired in transgenic cardiomyocytes: open probability and ensemble average currents were reduced by 60%. Furthermore, channels of transgenic myocytes were not stimulated by 8-Br-cAMP, in contrast to wild-types. Expression of malcomposed, dysfunctional L-type Ca 2+ channels in murine cardiomyocytes overexpressing hCa v1.2 explains the moderate enhancement of whole-cell currents and illustrates compensatory mechanisms in a transgenic disease model.