Marcos Rubio-Alarcón

Centro de Investigación Biomédica en Red

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Eva Delpón

Hospital General Universitario Gregorio Marañón

Anabel Cámara-Checa

Centro de Investigación Biomédica en Red

Ricardo Caballero

Centro de Investigación Biomédica en Red

Juan Tamargo

Universidad Complutense de Madrid

María Dago

Universidad Complutense de Madrid

Teresa Crespo-García

Universidad Complutense de Madrid

Josu Rapún

Centro de Investigación Biomédica en Red

Jorge Cebrián

Centro de Investigación en Red en Enfermedades Cardiovasculares

M. Luisa Marín

Universitat Politècnica de València

Paloma Nieto-Marín

Universidad Complutense de Madrid

Cooperative Institutions

Centro de Investigación en Red en Enfermedades Cardiovasculares

Centro de Investigación Biomédica en Red

Universidad Complutense de Madrid

Hospital General Universitario Gregorio Marañón

Instituto de Salud Carlos III

Universidad Autónoma de Madrid

Instituto de Investigación Biomédica de Salamanca

Complejo Hospitalario de Salamanca

Centro de Estudios Andaluces

Universidad de Salamanca

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Tbx5 variants disrupt Nav1.5 function differently in patients diagnosed with Brugada or Long QT Syndrome

Cardiovascular Research (2021)

Paloma Nieto-Marín David Tinaquero Raquel G. Utrilla Jorge Cebrián Andrés González-Guerra

Abstract Aims The transcription factor Tbx5 controls cardiogenesis and drives Scn5a expression in mice. We have identified two variants in TBX5 encoding p. D111Y and p. F206L Tbx5, respectively, in two unrelated patients with structurally normal hearts diagnosed with long QT (LQTS) and Brugada (BrS) syndrome. Here, we characterized the consequences of each variant to unravel the underlying disease mechanisms. Methods and results We combined clinical analysis with in vivo and in vitro electrophysiological and molecular techniques in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs), HL-1 cells, and cardiomyocytes from mice trans-expressing human wild-type (WT) or mutant proteins. Tbx5 increased transcription of SCN5A encoding cardiac Nav1.5 channels, while repressing CAMK2D and SPTBN4 genes encoding Ca/calmodulin kinase IIδ (CaMKIIδ) and βIV-spectrin, respectively. These effects significantly increased Na current (INa) in hiPSC-CMs and in cardiomyocytes from mice trans-expressing Tbx5. Consequently, action potential (AP) amplitudes increased and QRS interval narrowed in the mouse electrocardiogram. p. F206L Tbx5 bound to the SCN5A promoter failed to transactivate it, thus precluding the pro-transcriptional effect of WT Tbx5. Therefore, p. F206L markedly decreased INa in hiPSC-CM, HL-1 cells and mouse cardiomyocytes. The INa decrease in p. F206L trans-expressing mice translated into QRS widening and increased flecainide sensitivity. p. D111Y Tbx5 increased SCN5A expression but failed to repress CAMK2D and SPTBN4. The increased CaMKIIδ and βIV-spectrin significantly augmented the late component of INa (INaL) which, in turn, significantly prolonged AP duration in both hiPSC-CMs and mouse cardiomyocytes. Ranolazine, a selective INaL inhibitor, eliminated the QT and QTc intervals prolongation seen in p. D111Y trans-expressing mice. Conclusions In addition to peak INa, Tbx5 critically regulates INaL and the duration of repolarization in human cardiomyocytes. Our original results suggest that TBX5 variants associate with and modulate the intensity of the electrical phenotype in LQTS and BrS patients.

Nav1.5

Flecainide

10.1093/cvr/cvab045

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Citations (21)

Convergent activation of Ca ²⁺ permeability in two-pore channel 2 through distinct molecular routes

Science Signaling (2023)

Ryo� Saito Qianru Mu Yu Yuan Marcos Rubio-Alarcón Maria Eznarriaga

TPC2 is a pathophysiologically relevant lysosomal ion channel that is activated directly by the phosphoinositide PI(3,5)P2 and indirectly by the calcium ion (Ca2+)-mobilizing molecule NAADP through accessory proteins that associate with the channel. TPC2 toggles between PI(3,5)P2-induced, sodium ion (Na+)-selective and NAADP-induced, Ca2+-permeable states in response to these cues. To address the molecular basis of polymodal gating and ion-selectivity switching, we investigated the mechanism by which NAADP and its synthetic functional agonist, TPC2-A1-N, induced Ca2+ release through TPC2 in human cells. Whereas NAADP required the NAADP-binding proteins JPT2 and LSM12 to evoke endogenous calcium ion signals, TPC2-A1-N did not. Residues in TPC2 that bind to PI(3,5)P2 were required for channel activation by NAADP but not for activation by TPC2-A1-N. The cryptic voltage-sensing region of TPC2 was required for the actions of TPC2-A1-N and PI(3,5)P2 but not for those of NAADP. These data mechanistically distinguish natural and synthetic agonist action at TPC2 despite convergent effects on Ca2+ permeability and delineate a route for pharmacologically correcting impaired NAADP-evoked Ca2+ signals.

10.1126/scisignal.adg0661

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Zfhx3 Transcription Factor Represses the Expression of SCN5A Gene and Decreases Sodium Current Density (INa)

International Journal of Molecular Sciences (2021)

Marcos Rubio-Alarcón Anabel Cámara-Checa María Dago Teresa Crespo-García Paloma Nieto-Marín

The

10.3390/ijms222313031

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Zfhx3 decreases expression of cardiac Nav1.5 channels and inhibits sodium current

European Heart Journal (2021)

Ricardo Caballero Anabel Cámara-Checa Marcos Rubio-Alarcón Teresa Crespo-García María Dago

Abstract Background Zfhx3 (zinc finger homeobox 3) is a transcription factor (TF) encoded by the ZFHX3 gene. GWAS and gene-based association studies showed that ZFHX3 is one of the major atrial fibrillation (AF) susceptibility-conferring genes. The sodium current (INa), carried by Nav1.5 channels encoded by SCN5A, is responsible for atrial and ventricular action potential depolarization and determines cardiac excitability. Zfhx3 interacts with other TFs, such as Tbx5 and Pitx2c that increase and decrease INa, respectively. However, the effects of Zfhx3 on cardiac INa are currently unknown. Purpose We aimed to determine the effects of Zfhx3 on the INa on HL-1 cardiomyocytes. Methods cDNAs encoding human Zfhx3 together or not with Pitx2c or Tbx5 were transfected in HL-1 cells. Endogenous Zfhx3 expression in HL-1 cells was silenced by means of siRNAs. INa was recorded at room temperature using the whole-cell patch-clamp and luciferase reporter assays, qPCR and Western-blot (WB) analyses were also conducted. Results Expression analysis of RNA-seq data from human ventricular (n=432) samples (GTEx) demonstrated that Zfhx3 mRNA can be detected in the adult working myocardium. Transfection of Zfhx3 strongly reduced peak INa density (from −75.0±6.6 to −30.9±2.9 pA/pF; n≥26, P<0.001). In contrast, Zfhx3 silencing augmented INa density compared to cells transfected with scrambled siRNA (from −65.9±8.9 to −104.6±10.8 pA/pF; n≥8, P<0.05). Neither Zfhx3 expression nor silencing modified time and voltage dependence of activation and inactivation or the reactivation kinetics. Zfhx3 significantly reduced transcriptional activity of human SCN5A, PITX2 and TBX5 minimal promoters and, consequently, the mRNA and protein expression levels of Nav1.5, Pitx2c, and Tbx5 were diminished (n≥6, P<0.05). In cells transfected with Zfhx3 together with Pitx2c, but not with Tbx5, INa density was significantly smaller than in cells expressing WT Zfhx3 alone (n≥15, P<0.05). Further WB experiments demonstrated that Zfhx3 increased the expression of Nedd4–2 ubiquitin-protein ligase, which ubiquitinates Nav1.5 channels and favors their proteasomal degradation. Conclusions Zfhx3 inhibits INa as a result of a direct repressor effect on the SCN5A promoter, the modulation of Tbx5-increasing and Pitx2-decreasing effects on the INa, and the enhancement of Nav1.5 channel degradation. We propose a novel and complex mechanism that regulates the expression of sodium channels and the density of the INa, which are critical for the control of cardiac excitability. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministerio de Economía y CompetitividadComunidad Autόnoma de Madrid

10.1093/eurheartj/ehab724.3209

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Reply to Benndorff and DiFrancesco: Reliable human HCN4 single-channel recordings using the cell-attached configuration in expression systems

Proceedings of the National Academy of Sciences (2024)

Anabel Cámara-Checa Marcos Rubio-Alarcón María Dago Teresa Crespo-García Josu Rapún

Structures from the Stone Age can provide unique insights into Late Glacial and Mesolithic cultures around the Baltic Sea. Such structures, however, usually did not survive within the densely populated Central European subcontinent. Here, we ...The Baltic Sea basins, some of which only submerged in the mid-Holocene, preserve Stone Age structures that did not survive on land. Yet, the discovery of these features is challenging and requires cross-disciplinary approaches between archeology and ...

Mesolithic

Baltic sea

10.1073/pnas.2402992121

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Variable Penetrance and Expressivity of a Rare Pore Loss-of-Function Mutation (p.L889V) of Nav1.5 Channels in Three Spanish Families

International Journal of Molecular Sciences (2024)

María Gallego‐Delgado Anabel Cámara-Checa Marcos Rubio-Alarcón D.H. Heredero Jung Laura de la Fuente Blanco

A novel rare mutation in the pore region of Nav1.5 channels (p.L889V) has been found in three unrelated Spanish families that produces quite diverse phenotypic manifestations (Brugada syndrome, conduction disease, dilated cardiomyopathy, sinus node dysfunction, etc.) with variable penetrance among families. We clinically characterized the carriers and recorded the Na+ current (INa) generated by p.L889V and native (WT) Nav1.5 channels, alone or in combination, to obtain further insight into the genotypic–phenotypic relationships in patients carrying SCN5A mutations and in the molecular determinants of the Nav1.5 channel function. The variant produced a strong dominant negative effect (DNE) since the peak INa generated by p.L889V channels expressed in Chinese hamster ovary cells, either alone (−69.4 ± 9.0 pA/pF) or in combination with WT (−62.2 ± 14.6 pA/pF), was significantly (n ≥ 17, p < 0.05) reduced compared to that generated by WT channels alone (−199.1 ± 44.1 pA/pF). The mutation shifted the voltage dependence of channel activation and inactivation to depolarized potentials, did not modify the density of the late component of INa, slightly decreased the peak window current, accelerated the recovery from fast and slow inactivation, and slowed the induction kinetics of slow inactivation, decreasing the fraction of channels entering this inactivated state. The membrane expression of p.L889V channels was low, and in silico molecular experiments demonstrated profound alterations in the disposition of the pore region of the mutated channels. Despite the mutation producing a marked DNE and reduction in the INa and being located in a critical domain of the channel, its penetrance and expressivity are quite variable among the carriers. Our results reinforce the argument that the incomplete penetrance and phenotypic variability of SCN5A loss-of-function mutations are the result of a combination of multiple factors, making it difficult to predict their expressivity in the carriers despite the combination of clinical, genetic, and functional studies.

Penetrance

Nav1.5

Loss function

10.3390/ijms25094686

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A rare gain of function HCN4 gene mutation is responsible for inappropriate sinus tachycardia in a Spanish family

medRxiv (Cold Spring Harbor Laboratory) (2023)

Anabel Cámara-Checa Francesca Perín Marcos Rubio-Alarcón María Dago Teresa Crespo-García

ABSTRACT Background In a family with inappropriate sinus tachycardia (IST) we identified a novel mutation (p.V240M) of the hyperpolarization-activated cyclic nucleotide-gated type 4 (HCN4) channel, which contributes to the pacemaker current (I f ) in human sinoatrial node cells. Here we clinically study the family and functionally analyze the p.V240M variant. Methods Macroscopic (I HCN4 ) and single-channel currents were recorded using patch-clamp in cells expressing human native (WT) and/or p.V240M HCN4 channels. Results All p.V240M mutation carriers exhibited IST (mean heart rate 113[7] bpm, n=9), that in adults, was accompanied by cardiomyopathy. I HCN4 generated by p.V240M channels either alone or in combination with WT was significantly greater than that generated by WT channels. The variant, which lies in the N-terminal HCN domain, increased single-channel conductance and opening frequency and probability of HCN4 channels. Conversely, it did not modify channel sensitivity for cAMP and ivabradine or the level of expression at the membrane. Treatment with ivabradine based on functional data reversed the IST and the cardiomyopathy of the carriers. Conclusions The p.V240M gain-of-function variant increases I f during diastole, which explains the IST of the carriers. The results demonstrate the importance of the unique HCN domain in HCN4 which stabilizes the channels in the closed state. Funding Ministerio de Ciencia e Innovación (PID2020-118694RB-I00); Comunidad Autónoma de Madrid (P2022/BMD-7229), European Structural and Investment Funds); and Instituto de Salud Carlos III (CIBERCV; CB16/11/00303).

Ivabradine

10.1101/2023.01.20.23284606

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Regulation of cardiac ion channels by transcription factors: Looking for new opportunities of druggable targets for the treatment of arrhythmias

Biochemical Pharmacology (2022)

Teresa Crespo-García Anabel Cámara-Checa María Dago Marcos Rubio-Alarcón Josu Rapún

Cardiac electrical activity is governed by different ion channels that generate action potentials. Acquired or inherited abnormalities in the expression and/or function of ion channels usually result in electrophysiological changes that can cause cardiac arrhythmias. Transcription factors (TFs) control gene transcription by binding to specific DNA sequences adjacent to target genes. Linkage analysis, candidate-gene screening within families, and genome-wide association studies have linked rare and common genetic variants in the genes encoding TFs with genetically-determined cardiac arrhythmias. Besides its critical role in cardiac development, recent data demonstrated that they control cardiac electrical activity through the direct regulation of the expression and function of cardiac ion channels in adult hearts. This narrative review summarizes some studies showing functional data on regulation of the main human atrial and ventricular Na+, Ca2+, and K+ channels by cardiac TFs such as Pitx2c, Tbx20, Tbx5, Zfhx3, among others. The results have improved our understanding of the mechanisms regulating cardiac electrical activity and may open new avenues for therapeutic interventions in cardiac acquired or inherited arrhythmias through the identification of TFs as potential drug targets. Even though TFs have for a long time been considered as 'undruggable' targets, advances in structural biology have led to the identification of unique pockets in TFs amenable to be targeted with small-molecule drugs or peptides that are emerging as novel therapeutic drugs.

Druggability

Cardiac Electrophysiology

Cardiac action potential

10.1016/j.bcp.2022.115206

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Synthesis and Biological Assessment of 4,1-Benzothiazepines with Neuroprotective Activity on the Ca2+ Overload for the Treatment of Neurodegenerative Diseases and Stroke

Molecules (2021)

Lucía Viejo Marcos Rubio-Alarcón Raquel L. Arribas Manuel Moreno-Castro Raquel Pérez-Marín

In excitable cells, mitochondria play a key role in the regulation of the cytosolic Ca2+ levels. A dysregulation of the mitochondrial Ca2+ buffering machinery derives in serious pathologies, where neurodegenerative diseases highlight. Since the mitochondrial Na+/Ca2+ exchanger (NCLX) is the principal efflux pathway of Ca2+ to the cytosol, drugs capable of blocking NCLX have been proposed to act as neuroprotectants in neuronal damage scenarios exacerbated by Ca2+ overload. In our search of optimized NCLX blockers with augmented drug-likeness, we herein describe the synthesis and pharmacological characterization of new benzothiazepines analogues to the first-in-class NCLX blocker CGP37157 and its further derivative ITH12575, synthesized by our research group. As a result, we found two new compounds with an increased neuroprotective activity, neuronal Ca2+ regulatory activity and improved drug-likeness and pharmacokinetic properties, such as clog p or brain permeability, measured by PAMPA experiments.

Efflux

10.3390/molecules26154473

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Empagliflozin and dapagliflozin increase Na current in human cardiomyocytes

European Heart Journal (2022)

Ricardo Caballero María Dago Anabel Cámara-Checa Teresa Crespo-García Marcos Rubio-Alarcón

Abstract Background Empagliflozin (EMPA) and dapagliflozin (DAPA) are sodium-glucose cotransporter 2 inhibitors (SGLT2i) used for the treatment of type 2 Diabetes Mellitus (T2DM). Both drugs reduce morbidity and mortality in heart failure (HF) patients with reduced or preserved ejection fraction, even in the absence of T2DM. Moreover, these drugs decrease ventricular arrhythmias and sudden cardiac death in HF patients. The sodium current (INa), carried by Nav1.5 channels, is responsible for cardiac action potential (AP) depolarization and determines excitability and conduction velocity. In HF patients, the expression of Nav1.5 channels is reduced, leading to a decrease of ventricular excitability that enhances the arrhythmic risk. Purpose We aimed to determine the effects of EMPA and DAPA on human cardiac INa and AP characteristics. Methods Peak INa and ventricular-like APs were recorded in cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) using patch-clamp techniques. INa was also recorded in CHO cells transiently transfected with human Nav1.5+Navβ1 channels. In all cases, EMPA or DAPA (1 μM) were added to culture media and incubated for 24-h. Results APs recorded in hiPSC-CMs exhibited automatic activity and incubation with EMPA or DAPA did not modify spontaneous beating frequency (0.39±0.04 Hz; P>0.05, n≥16). In cells driven at 1 Hz, none of the drugs modified resting membrane potential (−76.7±1.4 mV; P>0.05, n≥11), but significantly increased AP amplitude from 98.6±3.6 to 105±2.2 (DAPA) and 107±2.3 mV (EMPA) (P<0.05). Interestingly, only EMPA lengthened AP duration measured at 20%, 50%, and 90% (from 605.6±31.3 to 760.5±59.0 ms, P<0.05) of repolarization. In hiPSC-CMs EMPA increased maximum INa density by 64% (from −156.0±28.0 to −256.4±28.1 pA/pF, P<0.05, n≥7) and shifted the midpoint (Vh) of the inactivation curve to more hyperpolarized potentials (from −97.3±4.5 to −108.6±4.4 mV, P<0.05, n≥7). In turn, DAPA increased maximum INa density by 24% (to −193.8±26.6 pA/pF) and shifted the Vh of the activation curve to more negative potentials (from −47.2±1.6 mV to −55.5±2.8 mV, P<0.05), an effect that would increase the INa at negative potentials coinciding with channel opening. None of the drugs modified the time course of current activation or inactivation. In CHO cells, EMPA and DAPA effects on INa were identical to those observed on hiPSC-CM. These results suggest that both SGLT2i increase INa by enhancing Nav1.5 expression into the cell membrane, by a direct gating effect on the channel, or by a combination of both. Conclusions In human cardiomyocytes, EMPA and DAPA increase INa and the AP amplitude. Moreover, EMPA, but not DAPA, prolonged AP duration. We propose that EMPA and DAPA exhibit a unique mechanism that increases cardiac excitability and conduction velocity and could contribute to the prevention of arrhythmic events in HF patients. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministerio de Ciencia e innovaciόnInstituto de de Salud Carlos III

Empagliflozin

Dapagliflozin

EMPA

Mexiletine

10.1093/eurheartj/ehac544.2885

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