Channels In Cardiomyocytes Research Articles

Atomistic mechanisms of the regulation of small-conductance Ca2+-activated K+ channel (SK2) by PIP2

Small-conductance Ca2+-activated K+ channels (SK, KCa2) are gated solely by intracellular microdomain Ca2+. The channel has emerged as a therapeutic target for cardiac arrhythmias. Calmodulin (CaM) interacts with the CaM binding domain (CaMBD) of the SK channels, serving as the obligatory Ca2+ sensor to gate the channels. In heterologous expression systems, phosphatidylinositol 4,5-bisphosphate (PIP2) coordinates with CaM in regulating SK channels. However, the roles and mechanisms of PIP2 in regulating SK channels in cardiomyocytes remain unknown. Here, optogenetics, magnetic nanoparticles, combined with Rosetta structural modeling, and molecular dynamics (MD) simulations revealed the atomistic mechanisms of how PIP2 works in concert with Ca2+-CaM in the SK channel activation. Our computational study affords evidence for the critical role of the amino acid residue R395 in the S6 transmembrane segment, which is localized in propinquity to the intracellular hydrophobic gate. This residue forms a salt bridge with residue E398 in the S6 transmembrane segment from the adjacent subunit. Both R395 and E398 are conserved in all known isoforms of SK channels. Our findings suggest that the binding of PIP2 to R395 residue disrupts the R395:E398 salt bridge, increasing the flexibility of the transmembrane segment S6 and the activation of the channel. Importantly, our findings serve as a platform for testing of structural-based drug designs for therapeutic inhibitors and activators of the SK channel family. The study is timely since inhibitors of SK channels are currently in clinical trials to treat atrial arrhythmias.

Crosstalk between PKA and PIAS3 regulates cardiac Kv4 channel SUMOylation

Post-translational SUMOylation of nuclear and cytosolic proteins maintains homeostasis in eukaryotic cells and orchestrates programmed responses to changes in metabolic demand or extracellular stimuli. In excitable cells, SUMOylation tunes the biophysical properties and trafficking of ion channels. Ion channel SUMOylation status is determined by the opposing enzyme activities of SUMO ligases and deconjugases. Phosphorylation also plays a permissive role in SUMOylation. SUMO deconjugases have been identified for several ion channels, but their corresponding E3 ligases remain unknown. This study shows PIAS3, a.k.a. KChAP, is a bona fide SUMO E3 ligase for Kv4.2 and HCN2 channels in HEK cells, and endogenous Kv4.2 and Kv4.3 channels in cardiomyocytes. PIAS3-mediated SUMOylation at Kv4.2-K579 increases channel surface expression through a rab11a-dependent recycling mechanism. PKA phosphorylation at Kv4.2-S552 reduces the current mediated by Kv4 channels in HEK293 cells, cardiomyocytes, and neurons. This study shows PKA mediated phosphorylation blocks Kv4.2-K579 SUMOylation in HEK cells and cardiomyocytes. Together, these data identify PIAS3 as a key downstream mediator in signaling cascades that control ion channel surface expression.

Eicosapentaenoic Acid Rescues Cav1.2-L-Type Ca2+ Channel Decline Caused by Saturated Fatty Acids via Both Free Fatty Acid Receptor 4-Dependent and -Independent Pathways in Cardiomyocytes.

Dietary intake of omega-3 polyunsaturated fatty acids (eicosapentaenoic acid, EPA) exerts antiarrhythmic effects, although the mechanisms are poorly understood. Here, we investigated the possible beneficial actions of EPA on saturated fatty acid-induced changes in the L-type Ca2+ channel in cardiomyocytes. Cardiomyocytes were cultured with an oleic acid/palmitic acid mixture (OAPA) in the presence or absence of EPA. Beating rate reduction in cardiomyocytes caused by OAPA were reversed by EPA. EPA also retrieved a reduction in Cav1.2 L-type Ca2+ current, mRNA, and protein caused by OAPA. Immunocytochemical analysis revealed a distinct downregulation of the Cav1.2 channel caused by OAPA with a concomitant decrease in the phosphorylated component of a transcription factor adenosine-3',5'-cyclic monophosphate (cAMP) response element binding protein (CREB) in the nucleus, which were rescued by EPA. A free fatty acid receptor 4 (FFAR4) agonist TUG-891 reversed expression of Cav1.2 and CREB mRNA caused by OAPA, whereas an FFAR4 antagonist AH-7614 abolished the effects of EPA. Excessive reactive oxygen species (ROS) accumulation caused by OAPA decreased Cav1.2 and CREB mRNA expressions, which was reversed by an ROS scavenger. Our data suggest that EPA rescues cellular Cav1.2-Ca2+ channel decline caused by OAPA lipotoxicity and oxidative stresses via both free fatty acid receptor 4-dependent and -independent pathways.

Dynamic Changes in Ion Channels during Myocardial Infarction and Therapeutic Challenges.

In different areas of the heart, action potential waveforms differ due to differences in the expressions of sodium, calcium, and potassium channels. One of the characteristics of myocardial infarction (MI) is an imbalance in oxygen supply and demand, leading to ion imbalance. After MI, the regulation and expression levels of K+, Ca2+, and Na+ ion channels in cardiomyocytes are altered, which affects the regularity of cardiac rhythm and leads to myocardial injury. Myocardial fibroblasts are the main effector cells in the process of MI repair. The ion channels of myocardial fibroblasts play an important role in the process of MI. At the same time, a large number of ion channels are expressed in immune cells, which play an important role by regulating the in- and outflow of ions to complete intracellular signal transduction. Ion channels are widely distributed in a variety of cells and are attractive targets for drug development. This article reviews the changes in different ion channels after MI and the therapeutic drugs for these channels. We analyze the complex molecular mechanisms behind myocardial ion channel regulation and the challenges in ion channel drug therapy.

Exploring translational approaches for fully biological cardiac rhythm restoration through ion channel gene therapy

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research Council (Starting grant 716509) to D.A. Pijnappels Background Recently it was demonstrated how the heart itself could be enabled to quickly restore its rhythm by realizing a biologically-integrated cardiac defibrillator (BioICD) through modification and subsequent expression of ion channels in cardiomyocytes [1]. By incorporating these frequency-dependent depolarizing ion channels, abnormal cardiac rhythm could be rapidly detected and terminated to restore sinus rhythm in a fully biological and shock-free manner. However, from a translational point of view, it remains unclear how such rhythm restoration can be realized via ion channel gene therapy. Purpose To explore and understand the importance of the distribution and number of BioICD-expressing cardiomyocytes in realizing fully biological restoration of cardiac rhythm. Methods To this purpose, two different realistic gene therapy configurations, i.e. those corresponding to systemic and local transgene delivery, were tested in a digital twin of human ventricular cardiac monolayers. In the systemic delivery group, BioICD-expressing cells were uniformly distributed over the tissue at different total expression percentages. In the local delivery group, circular areas were populated with BioICD-expressing cells, randomly arranged in a Gaussian distribution. Spiral waves were initiated in both groups and studied for 10 seconds. An additional set of simulations for the systemic delivery group involved an adapted BioICD ion channel with a slower opening and faster closing rate. Results Upon comparing both gene therapy methods, the systemic approach showed a gradually increasing arrhythmia termination chance with increasing BioICD-expression percentage, while local delivery did not result in termination. Instead, local delivery resulted in islands of ionic heterogeneity, causing attraction and anchoring of the spiral waves in a size and distance-dependent manner. Building on the results of the systemic approach, different ion channel parameters were tried, which resulted in normal rhythm being restored in all cases for >50% BioICD expressing cells. Time till termination was inversely related to the percentage, resulting in only 4.3s and 2.5s for 50% and 100%, respectively. Regarding termination, it was observed that conduction blocks appeared throughout the tissue and subsequently connected to force arrhythmic waves to terminate, while this process remained incomplete in the <50% groups. Conclusion This study reveals that wide-spread distribution of BioICD-expressing cardiomyocytes is required for the realization of fully biological self-restoration of cardiac rhythm, of which the efficiency is ion-channel-dynamics- and dosage-dependent. Local expression, however, results in stabilization of spiral wave activity. Further exploration of this emerging concept of biological cardioversion may not only expand our understanding of cardiac arrhythmias, but also pave the way to breakthrough advances in arrhythmia management.

The RGK protein rrad regulates cardiac calcium levels in zebrafish

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Science, Innovation and Universities, Spain Background/Introduction High voltage-activated Ca2+ (CaV) channels are essential for excitation-contraction coupling. RGK proteins (Gem, Rad, Rem, and Rem2) are small Ras-like G proteins that bind CaV β-subunits and profoundly inhibit CaV1/CaV2 channels. A recent study showed that Rad inhibits CaV1.2 channels in cardiomyocytes, thus mediating the enhanced inotropy and chronotropy by β-adrenergic agonists during the fight-or-flight response. The zebrafish has emerged as a useful in vivo animal model in cardiovascular physiology and pathophysiology. The role of zebrafish rrad, the orthologe of the human RAD, in the regulation of cardiac Ca2+ homeostasis in zebrafish has not been investigated. Purpose The aim of this study was to investigate the role of rrad in cardiac Ca2+ regulation in zebrafish. Methods We evaluated the cardiac Ca2+ dynamics, heart rhythm, and hemodynamics in transgenic zebrafish larvae that overexpress rrad in the heart, as well as in heterozygous rrad knockout larvae. Measurements were done in the beating heart of 3 dpf transgenic zebrafish larvae expressing the fluorescent Ca2+ biosensor Twitch-4. Results In some larvae, overexpression of rrad induced a severe enlargement of the atrium with pericardial edema and a high mortality rate. In addition, the ventricle displayed low Ca2+ levels and reduced contractility. The remaining larvae had no altered morphology, but the ventricular Ca2+ levels were reduced. On the other hand, preliminary results showed that down regulation of rrad in zebrafish larvae increased atrial and ventricular Ca2+ levels, like what was found in Rad knockout mice. Conclusion Taken together, these findings suggest that rrad in the zebrafish heart, as in mammals, is an inhibitor of the Ca2+ entry, presumably through CaV1/CaV2 channels, and demonstrate the zebrafish's utility as a model to study the involvement of rrad in cardiac function.

SGK1 inhibition normalizes action potential duration in transgenic LQT2 rabbits but not in LQT1, suggesting a novel gene-specific therapeutic approach in long QT syndrome

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): SNSF (Swiss National Science Fundation) Background Increasing evidence suggests that secondary remodeling in Na+ and Ca2+ homeostasis may further enhance the propensity to develop arrhythmias in K-channelopathies LQT1 and LQT2 syndrome. In this context, the Serum and Glucocorticoid regulated Kinase-1 (SGK-1) was recently identified as a major regulator of sodium channels in cardiomyocytes. It is mainly activated in pathological conditions, leading to increased INaLate, prolongation of action potential duration (APD) and increased susceptibility to ventricular arrhythmias, recapitulating LQTS phenotype. SGK1 inhibition might hence be a novel therapeutic target also in genetic LQTS subtypes. Methods In cardiomyocytes (CM) isolated from wild type (WT), LQT1 and LQT2 adult rabbits patch-clamp experiments were performed to record AP with SGK1-inhibitor(SGK1-inh, 3 µM) or DMSO vehicle control. Results In the perforated patch configuration at 37°C, 3 μM of SGK1-inh shortened APD90 in LQT2 CMs at 1 Hz (DMSO-control [ms]: 477.3 vs treated cells[ms]: 362.8 p < 0.0001) and at 2 Hz (DMSO-cells: 325.7 vs treated cells:295.4,p <0.05). In contrast, in WT and LQT1 CMs, no effect on APD90 was observed neither at 1 Hz (WT, DMSO-cells: 329.2 vs treated cells: 301.6 p=ns; LQT1, DMSO-cells: 429.8 vs treated cells: 431.6, p=ns) nor at 2 Hz (WT, DMSO-cells:256.8 vs treated cells:242 p=ns; LQT1,DMSO cells:310.4 vs treated cells: 315.8,p=ns). Importantly, the SGK1-inhibitor effect in LQT2 rabbit CMs was so pronounced that it normalized APD to the wildtype level (LQT2+SGK1-Inh: 362.8 vs WT:329.2, ps=ns). Furthermore, short-term-variability in APD was markedly reduced by SGK1-Inh in LQT2, indicating an antiarrhythmic effect. Conclusions SGK1-inhibition exerts a beneficial APD-shortening effect only in LQT2, indicating a gene-specific efficacy. The underlying mechanism is still unclear. We hypothesize that the SGK1 pathway/INaLate activation in LQT1 might not be as active as in LQT2. We are currently conducting more experiments on potential genotype differences in SGK1 activity and SGK1-mediated INaLate inhibition to further elucidate the differences.Effects of SGK1-inh on AP characteristic

Translational exploration of fully biological restoration of cardiac rhythm via ion channel gene therapy

Abstract Background Recently it was demonstrated how the heart itself could be enabled to quickly restore its rhythm by realizing a biologically-integrated cardiac defibrillator (BioICD) through modification and subsequent expression of ion channels in cardiomyocytes [1]. By incorporating these frequency-dependent depolarizing ion channels, abnormal cardiac rhythm could be rapidly detected and terminated to restore sinus rhythm in a fully biological and shock-free manner. However, from a translational point of view, it remains unclear how such rhythm restoration can be realized via ion channel gene therapy. Purpose To explore and understand the importance of the distribution and number of BioICD-expressing cardiomyocytes in realizing fully biological restoration of cardiac rhythm. Methods To this purpose, two different realistic gene therapy configurations, i.e. those corresponding to systemic and local transgene delivery, were tested in a digital twin of human ventricular cardiac monolayers. For the systemic delivery group, BioICD-expressing cells were homogeneously distributed over the tissue with fixed total expression percentage. For the local delivery group, circular areas were given BioICD-expressing cells, randomly patterned in a Gaussian distribution. In both groups spiral waves were initiated and subsequently studied for 10 seconds. For systemic delivery, an additional set of simulations was performed for an adapted BioICD ion channel with a slower opening and faster closing rate. Results Upon comparing both gene therapy methods, the systemic approach showed a gradually increasing arrhythmia termination chance with increasing BioICD-expression percentage, while local delivery did not result in termination. Instead, local delivery resulted in islands of ionic heterogeneity, causing attraction and anchoring of the spiral waves in a size and distance-dependent manner. Building on the results of the systemic approach, different ion channel parameters were tried, which resulted in normal rhythm being restored in all cases for >50% BioICD expressing cells. Time till termination was inversely related to the percentage, resulting in only 4.3s and 2.5s for 50% and 100%, respectively. Regarding termination, it was observed that conduction blocks appeared throughout the tissue and subsequently connected to force arrhythmic waves to terminate, while this process remained incomplete in the <50% groups. Conclusion This study reveals that wide-spread distribution of BioICD-expressing cardiomyocytes is required for the realization of fully biological self-restoration of cardiac rhythm, of which the efficiency is ion-channel-dynamics- and dosage-dependent. Local expression, however, results in stabilization of spiral wave activity. Further exploration of this emerging concept of biological cardioversion may not only expand our understanding of cardiac arrhythmias, but also pave the way to breakthrough advances in arrhythmia management.

The Therapeutic Potential of Four Main Compounds of Zanthoxylum nitidum (Roxb.) DC: A Comprehensive Study on Biological Processes, Anti-Inflammatory Effects, and Myocardial Toxicity.

Zanthoxylum nitidum (Roxb.) DC. (Z. nitidum) is a traditional Chinese medicinal plant that is indigenous to the southern regions of China. Previous research has provided evidence of the significant anti-inflammatory, antibacterial, and anticancer properties exhibited by Z. nitidum. The potential therapeutic effects and cardiac toxicity of Z. nitidum remain uncertain. The aim of this research was to investigate the potential therapeutic properties of the four main compounds of Z. nitidum in cardiovascular diseases, their impact on the electrical activity of cardiomyocytes, and the underlying mechanism of their anti-inflammatory effects. We selected the four compounds from Z. nitidum with a high concentration and specific biological activity: nitidine chloride (NC), chelerythrine chloride (CHE), magnoflorine chloride (MAG), and hesperidin (HE). A proteomic analysis was conducted on the myocardial tissues of beagle dogs following the administration of NC to investigate the role of NC in vivo and the associated biological processes. A bioinformatic analysis was used to predict the in vivo biological processes that MAG, CHE, and HE were involved in. Molecular docking was used to simulate the binding between compounds and their targets. The effect of the compounds on ion channels in cardiomyocytes was evaluated through a patch clamp experiment. Organ-on-a-chip (OOC) technology was developed to mimic the physiological conditions of the heart in vivo. Proteomic and bioinformatic analyses demonstrated that the four compounds of Z. nitidum are extensively involved in various cardiovascular-related biological pathways. The findings from the patch clamp experiments indicate that NC, CHE, MAG, and HE elicit a distinct activation or inhibition of the IK1 and ICa-L in cardiomyocytes. Finally, the anti-inflammatory effects of the compounds on cardiomyocytes were verified using OOC technology. NC, CHE, MAG, and HE demonstrate anti-inflammatory effects through their specific interactions with prostaglandin-endoperoxide synthase 2 (PTGS2) and significantly influence ion channels in cardiomyocytes. Our study provides a foundation for utilizing NC, CHE, MAG, and HE in the treatment of cardiovascular diseases.

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

FLRT3 and TGF-β/SMAD4 signalling: Impacts on apoptosis, autophagy and ion channels in supraventricular tachycardia.

To explore the underlying molecular mechanisms of supraventricular tachycardia (SVT), this study aimed to analyse the complex relationship between FLRT3 and TGF-β/SMAD4 signalling pathway, which affects Na+ and K+ channels in cardiomyocytes. Bioinformatics analysis was performed on 85 SVT samples and 15 healthy controls to screen overlapping genes from the key module and differentially expressed genes (DEGs). Expression profiling of overlapping genes, coupled with Receiver Operating Characteristic (ROC) curve analyses, identified FLRT3 as a hub gene. Invitro studies utilizing Ang II-stimulated H9C2 cardiomyocytes were undertaken to elucidate the consequences of FLRT3 silencing on cardiomyocyte apoptosis and autophagic processes. Utilizing a combination of techniques such as quantitative reverse-transcription polymerase chain reaction (qRT-PCR), western blotting (WB), flow cytometry, dual-luciferase reporter assays and chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR) assays were conducted to decipher the intricate interactions between FLRT3, the TGF-β/SMAD4 signalling cascade and ion channel gene expression. Six genes (AADAC, DSC3, FLRT3, SYT4, PRR9 and SERTM1) demonstrated reduced expression in SVT samples, each possessing significant clinical diagnostic potential. In H9C2 cardiomyocytes, FLRT3 silencing mitigated Ang II-induced apoptosis and modulated autophagy. With increasing TGF-β concentration, there was a dose-responsive decline in FLRT3 and SCN5A expression, while both KCNIP2 and KCND2 expressions were augmented. Moreover, a direct interaction between FLRT3 and SMAD4 was observed, and inhibition of SMAD4 expression resulted in increased FLRT3 expression. Our results demonstrated that the TGF-β/SMAD4 signalling pathway plays a critical role by regulating FLRT3 expression, with potential implications for ion channel function in SVT.

Identification of a novel Scn3b mutation in a Chinese Brugada syndrome pedigree: implications for Nav1.5 electrophysiological properties and intracellular distribution of Nav1.5 and Navβ3.

The Scn3b gene encodes for Navβ3, a pivotal regulatory subunit of the fast sodium channel in cardiomyocytes. However, its mutation status in the Chinese population suffering from Brugada Syndrome (BrS) has not been characterized, and the contributory pathophysiological mechanisms to disease pathology remain undefined. A Scn3b (c.260C>T, p.P87l) mutation was identified in a patient with BrS of Chinese descent. Functional analyses demonstrated that sodium channel activation for the wild type, mutant samples, and co-expression of both commenced at -55 mv and peaked at -25 mv. The mutant group exhibited a notable reduction, approximately 60%, in peak sodium channel activation current (INa) at -25 mv. The parameters for half-maximal activation voltages (V1/2) and slope factors (k) showed no significant differences when comparing wild type, mutant, and combined expression groups (P = 0.98 and P = 0.65, respectively). Additionally, no significant disparities were evident in terms of the steady-state sodium channel inactivation parameters V1/2 and k (with P-values of 0.85 and 0.25, respectively), nor were there significant differences in the activation time constant τ (P = 0.59) and late sodium current density (P = 0.23) across the wild-type, mutant, and co-expressed groups. Confocal imaging and Western blot analysis demonstrated decreased plasma membrane localization of SCN3B and SCN5A in the P87l group. Computational simulations of cardiac action potentials suggested that SCN3B P87l can alter the morphology of the action potentials within the endocardium and epicardium while reducing the peak of depolarization. The pathogenic impact of the Scn3b P87l mutation predominantly originates from a reduction in peak INa activation current coupled with decreased cell surface expression of Nav1.5 and Navβ3. These alterations may influence cardiac action potential configurations and contribute to the risk of ventricular arrhythmias in individuals with BrS.

In silico analysis of TRPM4 variants of unknown clinical significance

Background TRPM4 is a calcium-activated channel that selectively permeates monovalent cations. Genetic variants of the channel in cardiomyocytes are associated with various heart disorders, such as progressive familial heart block and Brugada syndrome. About97% of all known TRPM4 missense variants are classified as variants of unknown clinical significance (VUSs). The very large number of VUSs is a serious problem in diagnostics and treatment of inherited heart diseases. Methods and results We collected 233 benign or pathogenic missense variants in the superfamily of TRP channels from databases ClinVar, Humsavar and Ensembl Variation to compare performance of 22 algorithms that predict damaging variants. We found that ClinPred is the best-performing tool for TRP channels. We also used the paralogue annotation method to identify disease variants across the TRP family. In the set of 565 VUSs of hTRPM4, ClinPred predicted pathogenicity of 299 variants. Among these, 12 variants are also categorized as LP/P variants in at least one paralogue of hTRPM4. We further used the cryo-EM structure of hTRPM4 to find scores of contact pairs between parental (wild type) residues of VUSs for which ClinPred predicts a high probability of pathogenicity of variants for both contact partners. We propose that 68 respective missense VUSs are also likely pathogenic variants. Conclusions ClinPred outperformed other in-silico tools in predicting damaging variants of TRP channels. ClinPred, the paralogue annotation method, and analysis of residue contacts the hTRPM4 cryo-EM structure collectively suggest pathogenicity of 80 TRPM4 VUSs.

CDR1as promotes arrhythmias in myocardial infarction via targeting the NAMPT-NAD+ pathway

Cardiac ventricular arrhythmia triggered by acute myocardial infarction (AMI) is a major cause of sudden cardiac death. We have reported previously that an increased serum level of circular RNA CDR1as is a potential biomarker of AMI. However, the possible role of CDR1as in post-infarct arrhythmia remains unclear. This study in MI mice investigated the effects and underlying mechanism of CDR1as in ventricular arrhythmias associated with MI. We showed that knockdown of CDR1as abbreviated the duration of the abnormally prolonged QRS complex and QTc intervals and decreased susceptibility to ventricular arrhythmias. Optical mapping demonstrated knockdown of CDR1as also reduced post-infarct arrhythmia by increasing the conduction velocity and decreasing dispersion of repolarization. Mechanistically, CDR1as led to the depletion of NAD+ and caused mitochondrial dysfunction by directly targeting the NAMPT protein and repressing its expression. Moreover, CDR1as aggravated dysregulation of the NaV1.5 and Kir6.2 channels in cardiomyocytes, a change which was alleviated by the replenishment of NAD+. These findings suggest that anti-CDR1as is a potential therapeutic approach for ischemic arrhythmias.

Abstract P3148: Wnt/β-catenin Signaling Inhibits T-type Calcium Current And Causes Sinoatrial Node Dysfunction

Background: The T-type Ca 2+ channels (I Ca,T ) are expressed in the fetal and neonatal cardiomyocytes, but in the adult heart they are exclusively expressed in the cardiac conduction system, including the sinoatrial node (SAN). In contrast, the L-type Ca 2+ channels (I Ca,L ) are expressed ubiquitously in the adult heart and are important for cardiac contraction. The Wnt/β-catenin signaling (Wnt signaling) is activated in various heart disease and we and others have recently demonstrated that Wnt signaling inhibits cardiac Na + current. In the present study, we investigated if calcium channels are also regulated by Wnt signaling. Methods and Results: Because neonatal rat ventricular myocytes (NRVMs) express both I Ca,L and I Ca,T , we investigated the effect of Wnt signaling on NRVMs by treatment with either Wnt3a protein or CHIR-99021 (CHIR) (two different activators of the Wnt signaling). PCR array and RNA sequencing studies showed that Wnt signaling did not affect the mRNA of Cacna1c (encoding the α subunit of I Ca,L , Ca v 1.2), but the mRNA of Cacna1g (encoding the α subunit of I Ca,T , Ca v 3.1) was reduced in NRVMs (p<0.05, n=3) after treatment with either Wnt3a or CHIR. These observations were verified by qRT-PCR which showed dose-dependent reductions (p<0.05, n=4) in Cacna1g mRNA after CHIR treatment. Western blot and patch-clamp also demonstrated reductions (p<0.01, n=5) in channel protein and ionic current density of T-type, but not L-type, Ca 2+ channels after treatment with Wnt3a or CHIR. These observations suggest that Wnt signaling selectively inhibits T-type Ca 2+ channels in cardiomyocytes. Consistent with a critical role of T-type Ca 2+ channels in the pacemaker activity of SAN (the primary pacemaker of the heart), the spontaneous firing rates of isolated rat and mouse SAN tissues were reduced by ML-218 (an I Ca,T blocker) in a dose-dependent manner (p<0.01, n=5). In addition, activation of Wnt signaling in adult rat SAN tissues by CHIR led to slower and irregular beating, indicative of SAN dysfunction (p<0.01, n=5). Conclusions: These observations are consistent with the conclusions that Wnt/β-catenin signaling selectively inhibits T-type calcium current and may play a role in sinoatrial node dysfunction.

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.

PO-01-244 BMAL1 REGULATES THE CIRCADIAN RHYTHM OF RYR2 AND CONTRIBUTES TO CIRCADIAN VENTRICULAR ARRHYTHMIA IN CHRONIC HEART FAILURE AFTER MYOCARDIAL INFARCTION

Ventricular arrhythmia (VAs) is the major cause of sudden cardiac death in chronic heart failure (CHF) after myocardial infarction (MI), and previous studies have reported that VAs exist circadian oscillation in CHF. Peripheral circadian clock can regulate expression and function of ion channels in cardiomyocytes, which is associated with circadian rhythm of VAs in CHF. However, the regulation of peripheral circadian clock on Ryanodine Receptor 2 (RYR2), a calcium channel on cardiac sarcoplasmic reticulum, remain to be fully understood. We aimed to explore the regulation of peripheral circadian clock on RYR2 and VA events in CHF. CHF was induced by ligation of coronary artery of mice, and RT-PCR were performed to investigate the circadian rhythm of RYR2 in CHF myocardial tissue. Amplitude of calcium transient of ISO-induced cardiomyocytes at different time was measured to study the circadian rhythm of RYR2 function. To confirm the circadian oscillation of VAs, VAs incidences at different time of CHF wild type mice and cardiomyocyte-specific Bmal1 knockout (CKO) mice were investigated by programmed electrical stimulation. Infections of Ad-CLOCK and/or Ad-BMAL1 were applied to overexpress CLOCK, BMAL1 and CLOCK-BMAL1 in cardiomyocytes, and expression of RYR2 was measured by Western blot. Chromatin immunoprecipitation assay (ChIP) and luciferase (LUC) were applied to determine whether CLOCK-BMAL1 transcriptionally regulate RYR2 expression. CLOCK, BMAL1, and RYR2 exhibited notable circadian rhythm in CHF mice (Figure A, P<0.05). Amplitude of calcium transient at CT36 was lower than CT24 (Figure B, P<0.05). Compared to CT36, VAs had higher incidence at CT24(Figure C, P<0.05), and incidences of VAs had no significant difference between CT24 and CT36 in CKO Mice or using Dantrolene, an antagonist of RYR2. Ad-CLOCK and Ad-BMAL1 co-infection resulted in overexpression of RYR2 (Figure D, P<0.05), and BMAL1 bound to the enhancer of RYR2 gene and upregulated RYR2 expression transcriptionally (Figure E). In conclusion, BMAL1, as peripheral circadian clock, could contribute to circadian oscillation of VAs in CHF by regulating the circadian rhythm of RYR2.

SCN5A channelopathy: arrhythmia, cardiomyopathy, epilepsy and beyond.

Influx of sodium ions through voltage-gated sodium channels in cardiomyocytes is essential for proper electrical conduction within the heart. Both acquired conditions associated with sodium channel dysfunction (myocardial ischaemia, heart failure) as well as inherited disorders secondary to mutations in the gene SCN5A encoding for the cardiac sodium channel Nav1.5 are associated with life-threatening arrhythmias. Research in the last decade has uncovered the complex nature of Nav1.5 distribution, function, in particular within distinct subcellular subdomains of cardiomyocytes. Nav1.5-based channels furthermore display previously unrecognized non-electrogenic actions and may impact on cardiac structural integrity, leading to cardiomyopathy. Moreover, SCN5A and Nav1.5 are expressed in cell types other than cardiomyocytes as well as various extracardiac tissues, where their functional role in, e.g. epilepsy, gastrointestinal motility, cancer and the innate immune response is increasingly investigated and recognized. This review provides an overview of these novel insights and how they deepen our mechanistic knowledge on SCN5A channelopathies and Nav1.5 (dys)function. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Functional Regulation of KATP Channels and Mutant Insight Into Clinical Therapeutic Strategies in Cardiovascular Diseases

ATP-sensitive potassium channels (KATP channels) play pivotal roles in excitable cells and link cellular metabolism with membrane excitability. The action potential converts electricity into dynamics by ion channel-mediated ion exchange to generate systole, involved in every heartbeat. Activation of the KATP channel repolarizes the membrane potential and decreases early afterdepolarization (EAD)-mediated arrhythmias. KATP channels in cardiomyocytes have less function under physiological conditions but they open during severe and prolonged anoxia due to a reduced ATP/ADP ratio, lessening cellular excitability and thus preventing action potential generation and cell contraction. Small active molecules activate and enhance the opening of the KATP channel, which induces the repolarization of the membrane and decreases the occurrence of malignant arrhythmia. Accumulated evidence indicates that mutation of KATP channels deteriorates the regulatory roles in mutation-related diseases. However, patients with mutations in KATP channels still have no efficient treatment. Hence, in this study, we describe the role of KATP channels and subunits in angiocardiopathy, summarize the mutations of the KATP channels and the functional regulation of small active molecules in KATP channels, elucidate the potential mechanisms of mutant KATP channels and provide insight into clinical therapeutic strategies.

Mechanistic Insights Into Inflammation-Induced Arrhythmias: A Simulation Study.

Cardiovascular diseases are the primary cause of death of humans, and among these, ventricular arrhythmias are the most common cause of death. There is plausible evidence implicating inflammation in the etiology of ventricular fibrillation (VF). In the case of systemic inflammation caused by an overactive immune response, the induced inflammatory cytokines directly affect the function of ion channels in cardiomyocytes, leading to a prolonged action potential duration (APD). However, the mechanistic links between inflammatory cytokine-induced molecular and cellular influences and inflammation-associated ventricular arrhythmias need to be elucidated. The present study aimed to determine the potential impact of systemic inflammation on ventricular electrophysiology by means of multiscale virtual heart models. The experimental data on the ionic current of three major cytokines [i.e., tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1β), and interleukin-6 (IL-6)] were incorporated into the cell model, and the effects of each cytokine and their combined effect on the cell action potential (AP) were evaluated. Moreover, the integral effect of these cytokines on the conduction of excitation waves was also investigated in a tissue model. The simulation results suggested that inflammatory cytokines significantly prolonged APD, enhanced the transmural and regional repolarization heterogeneities that predispose to arrhythmias, and reduced the adaptability of ventricular tissue to fast heart rates. In addition, simulated pseudo-ECGs showed a prolonged QT interval—a manifestation consistent with clinical observations. In summary, the present study provides new insights into ventricular arrhythmias associated with inflammation.