Identification of N-terminal protein acetylation and arginine methylation of the voltage-gated sodium channel in end-stage heart failure human heart

The α subunit of the cardiac voltage-gated sodium channel, Naᵥ1.5, provides the rapid sodium inward current that initiates cardiomyocyte action potentials. Here, we analyzed for the first time the post-translational modifications of Naᵥ1.5 purified from end-stage heart failure human cardiac tissue. We identified R526 methylation as the major post-translational modification of any Naᵥ1.5 arginine or lysine residue. Unexpectedly, we found that the N terminus of Naᵥ1.5 was: 1) devoid of the initiation methionine, and 2) acetylated at the resulting initial alanine residue. This is the first evidence for N-terminal acetylation in any member of the voltage-gated ion channel superfamily. Our results open the door to explore Naᵥ1.5 N-terminal acetylation and arginine methylation levels as drivers or markers of end-stage heart failure

Do sodium channel proteolytic fragments regulate sodium channel expression?

© 2017 Taylor & Francis The cardiac voltage-gated sodium channel (gene: SCN5A, protein: Na V 1.5) is responsible for the sodium current that initiates the cardiomyocyte action potential. Research into the mechanisms of SCN5A gene expression has gained momentum over the last few years. We have recently described the transcriptional regulation of SCN5A by GATA4 transcription factor. In this addendum to our study, we report our observations that 1) the linker between domains I and II (L DI-DII ) of Na V 1.5 contains a nuclear localization signal (residues 474–481) that is necessary to localize L DI-DII into the nucleus, and 2) nuclear L DI-DII activates the SCN5A promoter in gene reporter assays using cardiac-like H9c2 cells. Given that voltage-gated sodium channels are known targets of proteases such as calpain, we speculate that Na V 1.5 degradation is signaled to the cell transcriptional machinery via nuclear localization of L DI-DII and subsequent stimulation of the SCN5A promoter

A Novel Missense Mutation, I890T, in the Pore Region of Cardiac Sodium Channel Causes Brugada Syndrome

Brugada syndrome (BrS) is a life-threatening, inherited arrhythmogenic syndrome associated with autosomal dominant mutations in SCN5A, the gene encoding the cardiac Na+ channel alpha subunit (Nav1.5). The aim of this work was to characterize the functional alterations caused by a novel SCN5A mutation, I890T, and thus establish whether this mutation is associated with BrS. The mutation was identified by direct sequencing of SCN5A from the proband's DNA. Wild-type (WT) or I890T Nav1.5 channels were heterologously expressed in human embryonic kidney cells. Sodium currents were studied using standard whole cell patch-clamp protocols and immunodetection experiments were performed using an antibody against human Nav1.5 channel. A marked decrease in current density was observed in cells expressing the I890T channel (from -52.0±6.5 pA/pF, n = 15 to -35.9±3.4 pA/pF, n = 22, at -20 mV, WT and I890T, respectively). Moreover, a positive shift of the activation curve was identified (V1/2 = -32.0±0.3 mV, n = 18, and -27.3±0.3 mV, n = 22, WT and I890T, respectively). No changes between WT and I890T currents were observed in steady-state inactivation, time course of inactivation, slow inactivation or recovery from inactivation parameters. Cell surface protein biotinylation analyses confirmed that Nav1.5 channel membrane expression levels were similar in WT and I890T cells. In summary, our data reveal that the I890T mutation, located within the pore of Nav1.5, causes an evident loss-of-function of the channel. Thus, the BrS phenotype observed in the proband is most likely due to this mutation. © 2013 Tarradas et al

Transcriptional regulation of the sodium channel gene (SCN5A) by GATA4 in human heart

Aberrant expression of the sodium channel gene (SCN5A) has been proposed to disrupt cardiac action potential and cause human cardiac arrhythmias, but the mechanisms of SCN5A gene regulation and dysregulation still remain largely unexplored. To gain insight into the transcriptional regulatory networks of SCN5A, we surveyed the promoter and first intronic regions of the SCN5A gene, predicting the presence of several binding sites for GATA transcription factors (TFs). Consistent with this prediction, chromatin immunoprecipitation (ChIP) and sequential ChIP (Re-ChIP) assays show co-occupancy of cardiac GATA TFs GATA4 and GATA5 on promoter and intron 1 SCN5A regions in freshfrozen human left ventricle samples. Gene reporter experiments show GATA4 and GATA5 synergism in the activation of the SCN5A promoter, and its dependence on predicted GATA binding sites. GATA4 and GATA6 mRNAs are robustly expressed in fresh-frozen human left ventricle samples as measured by highly sensitive droplet digital PCR (ddPCR). GATA5 mRNA is marginally but still clearly detected in the same samples. Importantly, GATA4 mRNA levels are strongly and positively correlated with SCN5A transcript levels in the human heart. Together, our findings uncover a novel mechanism of GATA TFs in the regulation of the SCN5A gene in human heart tissue. Our studies suggest that GATA5 but especially GATA4 are main contributors to SCN5A gene expression, thus providing a new paradigm of SCN5A expression regulation that may shed new light into the understanding of cardiac disease

Els factors GATA4 i GATA5 en la regulació transcripcional del gen que codifica pel canal de sodi cardíac (SCN5A)

The SCN5A gene encodes the alpha subunit of the cardiac sodium channel (NaV1.5), which is responsible for the influx of sodium ions through membrane of cardiomyocytes. Different evidences suggest that an aberrant expression of the SCN5A gene may cause cardiac arrhythmias. However, the mechanisms that control SCN5A expression regulation are largely unknown. This thesis proposes a new mechanism of SCN5A transcriptional regulation in the adult human heart: transcription factors GATA4 and GATA5 synergize in the activation of the SCN5A expression. In addition, it has been proposed that GATA4 activity on the SCN5A is regulated by acetylation/deacetylation via the acetyltransferase p300 and the deacetylase HDAC2. It has been identified three lysines of GATA4 that are targets of p300 and HDAC2. In summary, this study contributes to further understand the molecular basis of the cardiac arrhythmias associated with alteration of sodium currentsEl gen SCN5A codifica per la subunitat alfa del canal de sodi cardíac dependent de voltatge (NaV1.5), el qual permet l’entrada de ions sodi a través de la membrana dels cardiomiòcits. Diverses evidències suggereixen que una expressió anòmala del gen SCN5A pot donar lloc a arítmies cardíaques. Malauradament, els mecanismes que regulen l’expressió d’SCN5A són molt poc coneguts. Aquesta tesi proposa un nou mecanisme de regulació transcripcional del gen SCN5A en el cor humà adult: els factors de transcripció GATA4 i GATA5 activen sinèrgicament l’expressió del gen SCN5A. També s’ha proposat que l’activitat de GATA4 sobre SCN5A està regulada per un mecanisme d’acetilació/desacetilació a on hi participen l’acetiltransferasa p300 i la desacetilasa HDAC2. S’han identificat tres residus de lisines de GATA4 que són dianes de p300 i HDAC2. En resum, aquest estudi permet entendre millor les bases moleculars de les arítmies cardíaques associades amb alteracions del corrent de sod

Disseny d’un sistema orientat a la generació de signatures digitals per a circuits de senyal mixte

El projecte s’enquadra en el camp de test i diagnosi de circuits analògics i de senyal mixta. El test clàssic implica mesurar directament en el circuit les especificacions de disseny, però presenta dificultats per implementar-se de manera sistemàtica pel que resulten costos elevats. Una forma alternativa de dur a terme el test és mesurar altres paràmetres senzills del circuit i realitzar el test sobre aquests, entenent que hi ha una relació ben determinada entre les especificacions funcionals de disseny i dits paràmetres. L’objectiu del projecte és el disseny d’un sistema, que a partir de la caracterització d’aquests paràmetres de mesura, generi signatures digitals dels circuits a testejar. Així doncs, el present document engloba el disseny per a la captació i processat dels senyals analitzar. El sistema és capaç de captar 6 senyals analògics i fer-ne el processat digital (filtrat, mitjana i detecció d’extrems). Les sortides del sistema, digitals, proporcionen informació necessària per a prendre la decisió de test i/o diagnosi. El nucli del sistema és una FPGA (Field Programmable Gate Array), el dispositiu programable sobre el qual s’implementen els algorismes de tractament de dades. Finalment, la comprovació de la viabilitat del projecte s’efectua realitzant simulacions sobre el sistema dissenyat i comprovant que el comportament d’aquest acompleix les especificacions

Disseny d’un sistema orientat a la generació de signatures digitals per a circuits de senyal mixte

El projecte s’enquadra en el camp de test i diagnosi de circuits analògics i de senyal mixta. El test clàssic implica mesurar directament en el circuit les especificacions de disseny, però presenta dificultats per implementar-se de manera sistemàtica pel que resulten costos elevats. Una forma alternativa de dur a terme el test és mesurar altres paràmetres senzills del circuit i realitzar el test sobre aquests, entenent que hi ha una relació ben determinada entre les especificacions funcionals de disseny i dits paràmetres. L’objectiu del projecte és el disseny d’un sistema, que a partir de la caracterització d’aquests paràmetres de mesura, generi signatures digitals dels circuits a testejar. Així doncs, el present document engloba el disseny per a la captació i processat dels senyals analitzar. El sistema és capaç de captar 6 senyals analògics i fer-ne el processat digital (filtrat, mitjana i detecció d’extrems). Les sortides del sistema, digitals, proporcionen informació necessària per a prendre la decisió de test i/o diagnosi. El nucli del sistema és una FPGA (Field Programmable Gate Array), el dispositiu programable sobre el qual s’implementen els algorismes de tractament de dades. Finalment, la comprovació de la viabilitat del projecte s’efectua realitzant simulacions sobre el sistema dissenyat i comprovant que el comportament d’aquest acompleix les especificacions

A Missense Mutation in the Sodium Channel β2 Subunit Reveals SCN2B as a New Candidate Gene for Brugada Syndrome

Brugada Syndrome (BrS) is a familial disease associated with sudden cardiac death. A 20%-25% of BrS patients carry genetic defects that cause loss-of-function of the voltage-gated cardiac sodium channel. Thus, 70%-75% of patients remain without a genetic diagnosis. In this work, we identified a novel missense mutation (p.Asp211Gly) in the sodium β2 subunit encoded by SCN2B, in a woman diagnosed with BrS. We studied the sodium current (INa) from cells coexpressing Nav1.5 and wild-type (β2WT) or mutant (β2D211G) β2 subunits. Our electrophysiological analysis showed a 39.4% reduction in INa density when Nav1.5 was coexpressed with the β2D211G. Single channel analysis showed that the mutation did not affect the Nav1.5 unitary channel conductance. Instead, protein membrane detection experiments suggested that β2D211G decreases Nav1.5 cell surface expression. The effect of the mutant β2 subunit on the INa strongly suggests that SCN2B is a new candidate gene associated with BrS. Brugada Syndrome (BrS) is a familial disease associated mainly to a loss-of-function of the cardiac sodium channel. In this work, we identified a novel missense mutation (p.Asp211Gly) in the sodium β2 subunit encoded by SCN2B, in a woman diagnosed with BrS. Our electrophysiological analysis showed that the β2D211G provoked a reduction in INa density by decreasing Nav1.5 cell surface expression. These results strongly suggest that SCN2B is a new candidate gene for BrS. © 2013 WILEY PERIODICALS, INC

Transcriptional regulation of the sodium channel gene (SCN5A) by GATA4 in human heart

Aberrant expression of the sodium channel gene (SCN5A) has been proposed to disrupt cardiac action potential and cause human cardiac arrhythmias, but the mechanisms of SCN5A gene regulation and dysregulation still remain largely unexplored. To gain insight into the transcriptional regulatory networks of SCN5A, we surveyed the promoter and first intronic regions of the SCN5A gene, predicting the presence of several binding sites for GATA transcription factors (TFs). Consistent with this prediction, chromatin immunoprecipitation (ChIP) and sequential ChIP (Re-ChIP) assays show co-occupancy of cardiac GATA TFs GATA4 and GATA5 on promoter and intron 1 SCN5A regions in fresh-frozen human left ventricle samples. Gene reporter experiments show GATA4 and GATA5 synergism in the activation of the SCN5A promoter, and its dependence on predicted GATA binding sites. GATA4 and GATA6 mRNAs are robustly expressed in fresh-frozen human left ventricle samples as measured by highly sensitive droplet digital PCR (ddPCR). GATA5 mRNA is marginally but still clearly detected in the same samples. Importantly, GATA4 mRNA levels are strongly and positively correlated with SCN5A transcript levels in the human heart. Together, our findings uncover a novel mechanism of GATA TFs in the regulation of the SCN5A gene in human heart tissue. Our studies suggest that GATA5 but especially GATA4 are main contributors to SCN5A gene expression, thus providing a new paradigm of SCN5A expression regulation that may shed new light into the understanding of cardiac diseas

Biophysical parameters of WT and I890T channels.

<p>Activation and steady-state inactivation parameters were calculated by data fitting to Boltzmann functions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053220#s2" target="_blank">Methods</a>). <i>V_1/2</i> is the voltage for half-maximal activation or steady-state inactivation and <i>k</i> is the slope factor. Slow inactivation and recovery from inactivation data were fitted to mono-exponential functions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053220#s2" target="_blank">Methods</a>) to obtain the time constant <i>τ</i>. Values are expressed mean ± SE. *<i>p</i><0.05; **<i>p</i><0.01.</p