scholarly journals Functional Consequences of the SCN5A-p.Y1977N Mutation within the PY Ubiquitylation Motif: Discrepancy between HEK293 Cells and Transgenic Mice

2019 ◽  
Vol 20 (20) ◽  
pp. 5033 ◽  
Author(s):  
Simona Casini ◽  
Maxime Albesa ◽  
Zizun Wang ◽  
Vincent Portero ◽  
Daniela Ross-Kaschitza ◽  
...  
Keyword(s):  
Sudden Cardiac Death ◽  
Cardiac Death ◽  
Sodium Current ◽  
Hek293 Cells ◽  
Compensatory Mechanisms ◽  
Cardiac Sodium Channel ◽  
Functional Consequences ◽  
Py Motif ◽  
Type 3

Dysfunction of the cardiac sodium channel Nav1.5 (encoded by the SCN5A gene) is associated with arrhythmias and sudden cardiac death. SCN5A mutations associated with long QT syndrome type 3 (LQT3) lead to enhanced late sodium current and consequent action potential (AP) prolongation. Internalization and degradation of Nav1.5 is regulated by ubiquitylation, a post-translational mechanism that involves binding of the ubiquitin ligase Nedd4-2 to a proline-proline-serine-tyrosine sequence of Nav1.5, designated the PY-motif. We investigated the biophysical properties of the LQT3-associated SCN5A-p.Y1977N mutation located in the Nav1.5 PY-motif, both in HEK293 cells as well as in newly generated mice harboring the mouse homolog mutation Scn5a-p.Y1981N. We found that in HEK293 cells, the SCN5A-p.Y1977N mutation abolished the interaction between Nav1.5 and Nedd4-2, suppressed PY-motif-dependent ubiquitylation of Nav1.5, and consequently abrogated Nedd4-2 induced sodium current (INa) decrease. Nevertheless, homozygous mice harboring the Scn5a-p.Y1981N mutation showed no electrophysiological alterations nor changes in AP or (late) INa properties, questioning the in vivo relevance of the PY-motif. Our findings suggest the presence of compensatory mechanisms, with additional, as yet unknown, factors likely required to reduce the “ubiquitylation reserve” of Nav1.5. Future identification of such modulatory factors may identify potential triggers for arrhythmias and sudden cardiac death in the setting of LQT3 mutations.

scholarly journals Endurance exercise training normalizes repolarization and calcium-handling abnormalities, preventing ventricular fibrillation in a model of sudden cardiac death

2012 ◽  
Vol 113 (11) ◽  
pp. 1772-1783 ◽  
Author(s):  
Ingrid M. Bonilla ◽  
Andriy E. Belevych ◽  
Arun Sridhar ◽  
Yoshinori Nishijima ◽  
Hsiang-Ting Ho ◽  
...  
Keyword(s):  
Sudden Cardiac Death ◽  
Exercise Training ◽  
Endurance Exercise ◽  
Cardiac Death ◽  
Outward Current ◽  
Calcium Handling ◽  
Transient Outward Current ◽  
Cellular Electrophysiology ◽  
Endurance Exercise Training

The risk of sudden cardiac death is increased following myocardial infarction. Exercise training reduces arrhythmia susceptibility, but the mechanism is unknown. We used a canine model of sudden cardiac death (healed infarction, with ventricular tachyarrhythmias induced by an exercise plus ischemia test, VF+); we previously reported that endurance exercise training was antiarrhythmic in this model (Billman GE. Am J Physiol Heart Circ Physiol 297: H1171–H1193, 2009). A total of 41 VF+ animals were studied, after random assignment to 10 wk of endurance exercise training (EET; n = 21) or a matched sedentary period ( n = 20). Following (>1 wk) the final attempted arrhythmia induction, isolated myocytes were used to test the hypotheses that the endurance exercise-induced antiarrhythmic effects resulted from normalization of cellular electrophysiology and/or normalization of calcium handling. EET prevented VF and shortened in vivo repolarization ( P < 0.05). EET normalized action potential duration and variability compared with the sedentary group. EET resulted in a further decrement in transient outward current compared with the sedentary VF+ group ( P < 0.05). Sedentary VF+ dogs had a significant reduction in repolarizing K+ current, which was restored by exercise training ( P < 0.05). Compared with controls, myocytes from the sedentary VF+ group displayed calcium alternans, increased calcium spark frequency, and increased phosphorylation of S2814 on ryanodine receptor 2. These abnormalities in intracellular calcium handling were attenuated by exercise training ( P < 0.05). Exercise training prevented ischemically induced VF, in association with a combination of beneficial effects on cellular electrophysiology and calcium handling.

Abstract 1503: Knockdown of Mog1 Expression Reduces Sodium Currents by Reducing the Trafficking of Cardiac Sodium Channel Na V 1.5 to the Cell Membrane

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Susmita Chakrabarti ◽  
Sandro Yong ◽  
Shin Yoo ◽  
Ling Wu ◽  
Qing Kenneth Wang
Keyword(s):  
Sodium Channel ◽  
Ventricular Myocytes ◽  
Sodium Current ◽  
Expression System ◽  
Heart Association ◽  
Hek293 Cells ◽  
Similar Reduction ◽  
Mammalian Expression ◽  
Ventricular Cells ◽  
Cardiac Sodium Channel

The cardiac sodium channel (Na v 1.5) plays a significant role in cardiac physiology and leads to cardiac arrhythmias and sudden death when mutated. Modulation of Na v 1.5 activity can also arise from changes to accessory subunits or proteins. Our laboratory has recently reported that MOG1, a small protein that is highly conserved from yeast to humans, is a co-factor of Na v 1.5. Increased MOG1 expression has been shown to increase Na v 1.5 current density. In adult mouse ventricular myocytes, these two proteins were found to be co-localized at the intercalated discs. Here, we further characterize the regulatory role of MOG1 using the RNA interference technique. Sodium current was recorded in voltage-clamp mode from a holding potential of −100 mV and activated to −20 mV. In 3-day old mouse neonatal ventricular cells transfected with siRNA against mouse MOG1 decreased sodium current densities (pA/pF) compared to control or scramble siRNA treated cells (−10.2±3.3, n=11 vs. −165±16, n=20 or −117.9±11.7, n=11). A similar reduction in sodium current was observed in mammalian expression system consisting of HEK293 cells stably expressing human Na v 1.5, by transfecting siRNAs against either human or mouse MOG1 (−41.7±8.3, n=7 or, −82.6±9.6, n=7 vs. −130.6±11.5, n=7; −111.5±8.5, n=7, respectively). Immunocytochemistry revealed that the expression of MOG1 and Na v 1.5 were decreased in both HEK and neonatal cells when compared to scramble siRNAs or control groups. These results show that MOG1 is an essential co-factor for Na v 1.5 by way of a channel trafficking. Such interactions between MOG1 and Na v 1.5 suggest that early localization of MOG1 on the membrane of neonatal cardiomyocytes may be necessary for proper localization and the distribution of Na v 1.5 during cardiac development. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Common Ancestry-specific Ion Channel Variants Predispose to Drug-induced Arrhythmias

Circulation ◽  
2022 ◽  
Author(s):  
Yuko Wada ◽  
Tao Yang ◽  
Christian M. Shaffer ◽  
Laura L. Daniel ◽  
Andrew M. Glazer ◽  
...  
Keyword(s):  
Sodium Current ◽  
Common Ancestry ◽  
Drug Induced ◽  
Reference Allele ◽  
Qt Intervals ◽  
Cardiac Sodium Channel ◽  
Induced Pluripotent ◽  
Type 3 ◽  
Congenital Long Qt Syndrome

Background: Multiple reports associate the cardiac sodium channel gene ( SCN5A ) variants S1103Y and R1193Q with type 3 congenital long QT syndrome (LQTS) and drug-induced LQTS. These variants are, however, too common in ancestral populations to be highly arrhythmogenic at baseline: S1103Y allele frequency is 8.1% in Africans and R1193Q 6.1% in East Asians. R1193Q is known to increase late sodium current (I Na-L ) in cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) but the role of these variants in modulating repolarization remains poorly-understood. Methods: We determined the effect of S1103Y on QT intervals among Africans in a large electronic health record. Using iPSC-CMs carrying naturally occurring or genome-edited variants, we studied action potential durations (APDs) at baseline and after challenge with the repolarizing potassium current (I Kr ) blocker dofetilide, and I Na-L and I Kr at baseline. Results: In 1479 African subjects with no confounding medications or diagnoses of heart disease, QT in S1103Y carriers was no different from that in non-carriers. Similarly, baseline APD was no different in cells expressing the Y allele (SY, YY cells) compared to isogenic cells with the reference allele (SS cells). However, I Na-L was increased in SY and YY cells and the I Na-L blocker GS967 shortened APD in SY/YY but not SS cells (p<0.001). I Kr was increased almost 2-fold in SY/YY cells compared to SS cells (tail current: 0.66±0.1 vs 1.2±0.1 pA/pF, p<0.001). Dofetilide challenge prolonged APD at much lower concentrations in SY (4.1 nM [IQR 1.5-9.3], n=11) and YY (4.2 nM [1.7- 5.0], n=5) than in SS cells (249 nM [22.3-2905], n=14, p<0.001 and p<0.01, respectively) and elicited afterdepolarizations in 8/16 SY/YY cells but only in 1/14 SS cells. R1193Q cells similarly displayed no difference in baseline APD but increased I Kr and increased dofetilide sensitivity. Conclusions: These common ancestry-specific variants do not affect baseline repolarization, despite generating increased I Na-L . We propose that increased I Kr serves to maintain normal repolarization but increases the risk of manifest QT prolongation with I Kr block in variant carriers. Our findings further emphasize the need for inclusion of diverse populations in the study of adverse drug reactions.

Antiarrhythmic mechanisms during exercise

1996 ◽  
Vol 80 (6) ◽  
pp. 1853-1862 ◽  
Author(s):  
D. J. Paterson
Keyword(s):  
Sudden Cardiac Death ◽  
Cardiac Death ◽  
Protective Mechanism ◽  
Chemical Stress ◽  
Ionic Balance ◽  
Vigorous Exercise ◽  
Arterial Blood ◽  
Mutual Antagonism ◽  
Increased Risk

Exercise disturbs cardiac sympathovagal and ionic balance. In arterial blood, vigorous exercise can double plasma K(+), decrease pH by 0.4 unit, and raise catecholamines 15-fold. If any of these changes are experienced at rest, there is an increased risk of arrhythmia and cardiac arrest, yet in exercise they are usually tolerated. How the heart is protected from the chemical stress caused by exercise is not fully understood but may be related to a collective antiarrhythmic effect of these chemical changes, so when they combine there is a mutual antagonism. Catecholamines can offset the harmful cardiac effects of hyperkalemia and acidosis in isolated hearts and whole hearts in vivo and improve action-potential characteristics in K(+)-depolarized ventricular myocytes. This results from an increase in the inward Ca2(+) current that is modulated by both adrenergic and nonadrenergic hormones. Conversely, hyperkalemia can reduce or abolish the incidence of norepinephrine-induced arrhythmias. The efficacy of the mutual antagonism is reduced when the combination of acidosis, hyperkalemia, and high levels of norepinephrine are superimposed on a heart with regional ischemia or a small infarct. However, the heart may be at greatest risk in the postexercise period when plasma K(+) is low and the adrenergic tone is high. Little is known about this period, but abnormal regulation of electrolyte and cardiac sympathovagal balance may increase the incidence of arrhythmia, especially if there is underlying ischemia. Although regular physical activity can reduce the incidence of sudden cardiac death, recent epidemiological studies show that vigorous exercise can trigger myocardial infarction and sudden cardiac death, especially in habitually sedentary subjects with coronary artery disease. This may be partly related to disruption of the normal protective mechanism that allows the heart to cope with the chemical stress caused by exercise.

scholarly journals n-3 Fatty acids prevent whereas trans-fatty acids induce vascular inflammation and sudden cardiac death

2009 ◽  
Vol 102 (12) ◽  
pp. 1811-1819 ◽  
Author(s):  
Rafat A. Siddiqui ◽  
Kevin A. Harvey ◽  
Nargiz Ruzmetov ◽  
Steven J. Miller ◽  
Gary P. Zaloga
Keyword(s):  
Fatty Acids ◽  
Sudden Cardiac Death ◽  
Cardiac Death ◽  
Vascular Inflammation ◽  
Vascular Remodelling ◽  
Artery Ligation ◽  
Atherosclerotic Lesions ◽  
Collateral Growth

n-3 PUFA have well-recognised cardio-beneficial effects. In contrast, premature coronary deaths are associated with consumption of high levels of trans-fatty acids (TFA). The present study determined the effects of n-3 PUFA and TFA on sudden cardiac death and vascular inflammation. A rat coronary ligation model was used to study the effect of fatty acids on sudden cardiac death, whereas a mouse femoral artery ligation model was used to study compensatory vascular remodelling. Human aortic endothelial cells (HAEC) were utilised for the in vitro studies to investigate expression of inflammatory molecules. Feeding animals an n-3 PUFA-enriched diet caused a sevenfold increase in plasma n-3 PUFA compared with that of the TFA-fed group, whereas a TFA-enriched diet caused a 2·5-fold increase in plasma TFA compared with the n-3 PUFA group. Animals on a TFA diet had a lower survival rate due to sudden cardiac death and exhibited variable degrees of aortic atherosclerotic lesions. Animals on a TFA diet had diminished hindlimb collateral growth, whereas animals on the n-3 PUFA diet exhibited extensive collateral growth about ligated regions. HAEC treated with TFA (trans-18 : 2) showed significantly increased expression of intracellular adhesion molecule-1 and nitrosylation of cellular proteins than those treated with DHA (n-3 PUFA, 22 : 6). The in vivo study demonstrates that, in contrast to TFA, n-3 PUFA improve animal survival after myocardial infarction, prevent development of atherosclerotic lesions and stimulate compensatory vascular remodelling. The in vitro study demonstrates that TFA induce, while n-3 PUFA prevent, vascular inflammation.

scholarly journals MOG1 restores the expression and function of SCN5A-p.R104W through sec23a-mediated forward trafficking

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
L Luo ◽  
Y Wang ◽  
Y Du ◽  
C Dong ◽  
A Ma ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Sodium Current ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Funding Source ◽  
Inherited Disease ◽  
Patch Clamp Recording ◽  
Knock Out ◽  
Cardiac Sodium Channel ◽  
And Function

Abstract Background Brugada syndrome (BrS) is an inherited disease which causes fatal arrhythmias and sudden cardiac death. Mutations in SCN5A gene, which encoding cardiac sodium channel (NaV1.5), are the most common genotype of BrS patients. Some SCN5A-related variants were reported to retain NaV1.5 in endoplasmic reticulum (ER) due to trafficking deficiency. MOG1 was previously reported to interact with NaV1.5 and increased sodium current (INa) through enhancing the trafficking. However, its molecular mechanisms are still unclear. Coat protein complex II (COPII) is responsible for the ER to Golgi transport. Sec23 forms the inner coat of COPII and participates in cargo proteins selection. Purpose To demonstrate that MOG1 rescues SCN5A-related variants by enhancing the forward trafficking through Sec23a-NaV1.5 interaction. Methods Site directed mutagenesis, immunofluorescence staining, biotinylation assay, Western blot analysis and whole-cell patch clamp recording were used. CRISPR/Cas9 was used to knock out Sec23a expression in HEK293 cells. Results We found that SCN5A-p.R104W was characterized as reduced NaV1.5 level and lack of INa. The variant SCN5A-p.R104W was mainly distributed in ER. MOG1 could rescue the total and surface expression of SCN5A-p.R104W but could not restore INa (Figure 1a). Considering that most patients are heterozygous, co-transfection of SCN5A-WT and SCN5A-p.R104W were obtained. We found MOG1 could increase both NaV1.5 level and INa of heterozygous expressed SCN5A-p.R104W. We further revealed an interaction between NaV1.5 and Sec23a by co-immunoprecipitation (Co-IP) assay. The interaction between NaV1.5 and Sec23a was increased by MOG1, which indicates that Sec23a participates in MOG1-mediated increase in NaV1.5 level (Figure 1b). Knockout of Sec23a reduced cell surface, but not total, NaV1.5 level (Figure 1c and 1d). Next, the Sec23a knockout HEK293 cells were co-transfected with SCN5A-p.R104W and pcDNA3 or MOG1. MOG1 could not increase SCN5A-p.R104W protein level in Sec23a knockout cells. Conclusion Our data demonstrated a novel mechanism that MOG1 restores the expression and function of SCN5A-p.R104W by enhancing its forward trafficking through Sec23a-NaV1.5 interaction. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Natural Science Foundation of China

scholarly journals Cardiac Sodium Channel Gene Variants and Sudden Cardiac Death in Women

Circulation ◽  
2008 ◽  
Vol 117 (1) ◽  
pp. 16-23 ◽  
Author(s):  
Christine M. Albert ◽  
Edwin G. Nam ◽  
Eric B. Rimm ◽  
Hong Wei Jin ◽  
Roger J. Hajjar ◽  
...  
Keyword(s):  
Sudden Cardiac Death ◽  
Sodium Channel ◽  
Cardiac Death ◽  
Gene Variants ◽  
Channel Gene ◽  
Sodium Channel Gene ◽  
Cardiac Sodium Channel

Effect of β-adrenergic blockade on dynamic electrical restitution in vivo

2004 ◽  
Vol 287 (1) ◽  
pp. H390-H394 ◽  
Author(s):  
Steven C. Hao ◽  
David J. Christini ◽  
Kenneth M. Stein ◽  
Peter N. Jordan ◽  
Sei Iwai ◽  
...  
Keyword(s):  
Sudden Cardiac Death ◽  
Action Potential ◽  
Right Ventricular ◽  
Cardiac Death ◽  
Monophasic Action Potential ◽  
Adrenergic Blockade ◽  
Arrhythmogenic Substrate ◽  
Restitution Curve ◽  
Β Blockers

The slope of the action potential duration (APD) restitution curve may be a significant determinant of the propensity to develop ventricular fibrillation, with steeper slopes associated with a more arrhythmogenic substrate. We hypothesized that one mechanism by which β-blockers reduce sudden cardiac death is by flattening the APD restitution curve. Therefore, we investigated whether infusion of esmolol modulates the APD restitution curve in vivo. In 10 Yorkshire pigs, dynamic APD restitution curves were determined from measurements of APD at 90% repolarization with a monophasic action potential catheter positioned against the right ventricular septum during right ventricular apical pacing in the basal state and during infusion of esmolol. APD restitution curves were fitted to the three-parameter ( a, b, c) exponential equation, APD = a·[1 − e(− b·DI)] + c, where DI is the diastolic interval. Esmolol decreased the maximal APD slope, 0.68 ± 0.14 vs. 0.94 ± 0.24 (baseline), P = 0.002, and flattened the APD restitution curve at shorter DIs, 75 and 100 ms ( P < 0.05). To compare the slopes of the APD restitution curves at similar steady states, slopes were also computed at points of intersection between the restitution curve and the lines representing pacing at a fixed cycle length (CL) of 200, 225, 250, 275, and 300 ms using the relationship CL = APD + DI. Esmolol decreased APD restitution slopes at CLs 200–275 ms ( P < 0.05). Esmolol flattens the cardiac APD restitution curve in vivo, particularly at shorter CLs and DIs. This may represent a novel mechanism by which β-blockers prevent sudden cardiac death.

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