GPD1-L is a novel gene that may affect trafficking of the cardiac Na+ channel to the cell surface. A GPD1-L mutation decreases SCN5A surface membrane expression, reduces inward Na+ current, and causes Brugada syndrome.

Rationale: Mutations in glycerol-3-phosphate dehydrogenase 1-like (GPD1-L) protein reduce cardiac Na ؉ current (I Na ) and cause Brugada Syndrome (BrS). GPD1-L has >80% amino acid homology with glycerol-3-phosphate dehydrogenase, which is involved in NAD-dependent energy metabolism.Objective: Therefore, we tested whether NAD(H) could regulate human cardiac sodium channels (Na v 1.5).

Abstract-Heart failure (HF) is associated with reduced cardiac Na ϩ channel (SCN5A) current. We hypothesized that abnormal transcriptional regulation of this ion channel during HF could help explain the reduced current. Using human hearts explanted at the transplantation, we have identified 3 human C-terminal SCN5A mRNA splicing variants predicted to result in truncated, nonfunctional channels. As compared with normal hearts, the explanted ventricles showed an upregulation of 2 of the variants and a downregulation of the full-length mRNA transcript such that the E28A transcript represented only 48.5% (PϽ0.01) of the total SCN5A mRNA. This correlated with a 62.8% (PϽ0.01) reduction in Na ϩ channel protein. Lymphoblasts and skeletal muscle expressing SCN5A also showed identical C-terminal splicing variants. Variants showed reduced membrane protein and no functional current. Transfection of truncation variants into a cell line stably transfected with the full-length Na ϩ channel resulted in dose-dependent reductions in channel mRNA and current. Introduction of a premature truncation in the C-terminal region in a single allele of the mouse SCN5A resulted in embryonic lethality. Embryonic stem cell-derived cardiomyocytes expressing the construct showed reductions in Na ϩ channel-dependent electrophysiological parameters, suggesting that the presence of truncated Na ϩ channel mRNA at levels seen in HF is likely to be physiologically significant. In summary, chronic HF was associated with an increase in 2 truncated SCN5A variants and a decrease in the native mRNA. These splice variations may help explain a loss of Na Key Words: sodium channels Ⅲ transcriptional regulation Ⅲ mRNA splice variations Ⅲ heart failure Ⅲ arrhythmia H uman heart failure (HF) is associated with decreased cardiac voltage-gated sodium channel current. 1,2 Genetically mediated decreases in Na ϩ current have been implicated in the risk for sudden death, [3][4][5] and Na ϩ channel changes may contribute to the increased risk of sudden death in HF. 6,7 Because transcriptional alterations in other ion channels have been noted to contribute to current changes in HF, 8,9 we investigated Na ϩ channel protein and mRNA abundance in hearts explanted during cardiac transplantation to determine whether there were changes that might explain the reduced Na ϩ current previously reported in this tissue. Materials and Methods Detection of Human SCN5A 3UTR Variants by Rapid Amplification of cDNA Ends PCRTotal human RNA from normal fetal and adult whole hearts was purchased from Clontech (Mountain View, Calif). The RNA ligasemediated rapid amplification of cDNA ends (RACE) method was used to characterize the 3Ј ends of the human SCN5A mRNA using the GeneRacer kit (Invitrogen, Carlsbad, Calif). Primary and nested PCR reactions were performed with primers HE26F (on exon 26) and HE27F (on exon 27) specific to the human SCN5A gene and the GeneRacer 3Ј primer for amplifying the 3Ј-end fragment. The nested PCR products were cloned into pCR4-TOPO vector (Invitrogen) and se...

(ANG II) increases oxidative stress and is associated with increased risk of sudden cardiac death. The cardiac Na ϩ channel promoter contains elements that confer redox sensitivity. We tested the hypothesis that ANG IImediated oxidative stress may modulate Na ϩ channel current through altering channel transcription. In H9c2 myocytes treated for 48 h with ANG II (100 nmol/l) or H2O2 (10 mol/l) showed delayed macroscopic inactivation, increased late current, and 59.6% and 53.8% reductions in Na ϩ current, respectively (P Յ 0.01). By quantitative real-time RT-PCR, the cardiac Na ϩ channel (scn5a) mRNA abundance declined by 47.3% (P Ͻ 0.01) in H9c2 myocytes treated for 48 h with 100 nmol/l ANG II. A similar change occurred with 20 mol/l H2O2 (46.9%, P Ͻ 0.01) after 48 h. Comparable effects were seen in acutely isolated ventricular myocytes. The effects of ANG II could be inhibited by prior treatment of H9c2 cells with scavengers of reactive oxygen species or an inhibitor of the NADPH oxidase. Mutation of the scn5a promoter NF-B binding site prevented decreased activity in response to ANG II and H2O2. Gel shift and chromosomal immunoprecipitation assays confirmed that nuclear factor (NF)-B bound to the scn5a promoter in response to ANG II and H2O2. Overexpression of the p50 subunit of NF-B in H9c2 cells reduced scn5a mRNA (77.3%, P Ͻ 0.01). In conclusion, ANG II can decrease scn5a transcription and current. This effect appears to be through production of H2O2 resulting in NF-B binding to the Na ϩ channel promoter. arrhythmia, gene expression; sodium channel; redox signaling; renin angiotensin system ACTIVATION of the renin-angiotensin system (RAS) has been implicated in arrhythmia associated with heart failure because inhibitors of this pathway reduce the incidence of sudden death (18,39,42,43). One major effecter of RAS activation is angiotensin II (ANG II), produced by enzymatic activity of angiotensin-converting enzyme (ACE) on angiotensin I. ANG II is known to increase oxidative stress through NADPH oxidase activation (7,20,31). Increased oxygen free radical production is associated with congestive heart failure (13, 21) and arrhythmias (6, 32). Nevertheless, the molecular basis whereby ANG II may cause arrhythmias and any role for ANG II-induced oxidative stress are not clear.Ion channel transcriptional regulation is implicated in increasing ventricular and atrial arrhythmic risk (1,9,25,29,44).Often referred to as electrical remodeling, the changes in myocyte electrical properties in states of increased arrhythmic risk are related to underlying changes in expression of several ion channel genes, including reductions in connexins and Na ϩ channels, and may be responsible for the arrhythmic effects of ANG II. Downregulations of Na ϩ channels and connexin 43 are seen in heart failure, a condition associated with increased RAS activation (4, 15, 37, 46). Moreover, forms of electrical remodeling can be inhibited by agents altering RAS signaling and by antioxidants (8,24,38), suggesting that ANG IImediated ion channe...

The SCN5A gene encodes a voltage-sensitive sodium channel expressed in cardiac and skeletal muscle. Coding region mutations cause cardiac sudden death syndromes and conduction system failure. Polymorphisms in the 5-sequence adjacent to the SCN5A gene have been linked to cardiac arrhythmias. We identified three alternative 5-splice variants (1A, 1B, and 1C) of the untranslated exon 1 and two 3-variants in the murine Scn5a mRNA. Two of the exon 1 isoforms (1B and 1C) were novel when compared with the published human and rat SCN5A sequences. Quantitative real time PCR results showed that the abundance of the isoforms varied during cardiac development. The 1A, 1B, and 1C mRNA splice variants increased 7.8 ؎ 1.7-fold (E1A), 6.0 ؎ 1.0-fold (E1B), and 20.6 ؎ 3.7-fold (E1C) from fetal to adult heart, respectively. Promoter deletion and luciferase reporter gene analysis using cardiac and skeletal muscle cell lines demonstrated a pattern of distinct cardiac-specific enhancer elements associated with exons 1A and 1C. In the case of exon 1C, the enhancer element appeared to be within the exon. A 5-repressor preceded each cardiac enhancer element. We concluded that the murine Na ؉ channel has both 5-and 3-untranslated region mRNA variants that are developmentally regulated and that the promoter region contains two distinct cardiac-specific enhancer regions. The presence of homologous human splicing suggests that that these regions may be fruitful new areas of study in understanding cardiac sodium channel regulation and the genetic susceptibility to sudden death.

(RAS) system activation is associated with an increased risk of sudden death. Previously, we used cardiac-restricted angiotensin-converting enzyme (ACE) overexpression to construct a mouse model of RAS activation. These ACE 8/8 mice die prematurely and abruptly. Here, we have investigated cardiac electrophysiological abnormalities that may contribute to early mortality in this model. In ACE 8/8 mice, surface ECG voltages are reduced. Intracardiac electrograms showed atrial and ventricular potential amplitudes of 11% and 24% compared with matched wild-type (WT) controls. The atrioventricular (AV), atrioHisian (AH), and Hisian-ventricular (HV) intervals were prolonged 2.8-, 2.6-, and 3.9-fold, respectively, in ACE 8/8 vs. WT mice. Various degrees of AV nodal block were present only in ACE 8/8 mice. Intracardiac electrophysiology studies demonstrated that WT and heterozygote (HZ) mice were noninducible, whereas 83% of ACE 8/8 mice demonstrated ventricular tachycardia with burst pacing. Atrial connexin 40 (Cx40) and connexin 43 (Cx43) protein levels, ventricular Cx43 protein level, atrial and ventricular Cx40 mRNA abundances, ventricular Cx43 mRNA abundance, and atrial and ventricular cardiac Na ϩ channel (Scn5a) mRNA abundances were reduced in ACE 8/8 compared with WT mice. ACE 8/8 mice demonstrated ventricular Cx43 dephosphorylation. Atrial and ventricular L-type Ca 2ϩ channel, Kv4.2 K ϩ channel ␣-subunit, and Cx45 mRNA abundances and the peak ventricular Na ϩ current did not differ between the groups. In isolated heart preparations, a connexin blocker, 1-heptanol (0.5 mM), produced an electrophysiological phenotype similar to that seen in ACE 8/8 mice. Therefore, cardiac-specific ACE overexpression resulted in changes in connexins consistent with the phenotype of low-voltage electrical activity, conduction defects, and induced ventricular arrhythmia. These results may help explain the increased risk of arrhythmia in states of RAS activation such as heart failure.peptidyl-dipeptidase A; angiotensin II; heart block ARRHYTHMIC SUDDEN DEATH is a common terminal event in various cardiomyopathies and end-stage heart failure. Upregulation of the renin-angiotensin system (RAS) has been implicated in risk of sudden death in these conditions. A critical component of this system is angiotensin-converting enzyme (ACE), which produces the eight-amino acid peptide angiotensin II (ANG II), a major effector peptide of the RAS. In humans, increased ANG II levels are associated with an increased risk of arrhythmia (2), which is reduced by use of ACE inhibitors or ANG II receptor blockers (4,13,20,23,27,30,49).A number of ion-handling protein changes have been posited to underlie the increase in risk of arrhythmia in states of RAS activation, and ANG II is known to act on a number of these proteins (3, 41). For example, ANG II has been implicated in Na ϩ -K ϩ pump regulation (24). Furthermore, ANG II inhibits the Ca 2ϩ -activated K ϩ current in vascular smooth muscle cells (51) and the delayed rectifier K ϩ currents in heart and...

BACKGROUND-Brugada and Long QT type 3 syndromes are linked to sodium channel mutations and clinically cause arrhythmias that lead to sudden death. We have identified a novel threonine to isoleucine missense mutation at position 353 (T353I) adjacent to the pore-lining region of domain I of the cardiac sodium channel (SCN5a) in family with Brugada syndrome. Both male and female carriers are symptomatic at young ages, have typical Brugada-type ECG changes, and have relatively normal corrected QT intervals.

We examine the Hoxc12 RNA expression pattern during both hair follicle morphogenesis and cycling in direct comparison to its only upstream neighbor, Hoxc13. Expression of both genes is restricted to the epidermal part of the follicle excluding the outer root sheath and interfollicular epidermis in a distinct stage-dependent and cyclical manner. During the progressive growth phase (anagen) of developing and cycling follicles, the distinct proximo-distal expression domain of Hoxc12 overlaps only proximally, at the upper-most region of the bulb, with the more proximally restricted Hoxc13 domain. This arrangement of the expression domains of the two genes along the proximal-toward-distal axis of increasing follicular differentiation correlates with the sequential expression of first Hoxc13 and then Hoxc12. This indicates a reversal of the typical temporal colinearity of Hox gene activation otherwise observed along the anterior-posterior morphogenetic axis of the embryo (review: Cell 78 (1994) 191).