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Carmen R Valdivia

Publications and source records attributed to Carmen R Valdivia.

11 recordsLinked to original sources

Partial expression defect for the SCN5A missense mutation G1406R depends on splice variant background Q1077 and rescue by mexiletine.

Mutations in the cardiac Na(+) channel gene SCN5A cause loss of function and underlie arrhythmia syndromes. SCN5A in humans has two splice variants, one lacking a glutamine at position 1077 (Q1077del) and one containing Q1077. We investigated the effect of splice variant background on loss of function and rescue for G1406R, a mutation reported to cause Brugada syndrome. Mutant and wild-type (WT) channels in both backgrounds were transfected into HEK-293 cells and incubated for up to 72 h with and without mexiletine. At 8 h, neither current nor cell surface expression was observed for the mutant in either background, but both were present in WT channels. At 24 h, small (<10% compared with WT) currents were noted and accompanied by cell surface expression. At 48 h, current density was approximately 40% of WT channels for the mutant in the Q1077del variant background but remained at <10% of WT channels in Q1077. Current levels were stable by 72 h. Coexpression with beta(1)- or beta(3)-subunits or insertion of the polymorphism H558R in the background did not significantly affect current expression. Mexiletine restored current density of the mutant channel in both backgrounds to nearly WT levels. The mutant channels also showed a negative shift in inactivation, slower recovery, and enhanced slow inactivation, consistent with a loss of function phenotype. These data show that a trafficking defect may be partial and time dependent and may differ with the splice variant background. Also, expression defects and gating abnormalities may contribute to loss of function for the same mutation.

Alternative Splicing↗

Ranolazine and late cardiac sodium current--a therapeutic target for angina, arrhythmia and more?

Ranolazine is a new antianginal drug approved for clinical use in the United States in January 2006. A study published in this same issue of the British Journal of Pharmacology characterizes ranolazine block of late sodium current caused by the long QT syndrome 3 mutations. This commentary discusses the implications of that study and the background and implications for block of late cardiac sodium current in general.

Acetanilides↗

Specific serine proteases selectively damage KCNH2 (hERG1) potassium channels and I(Kr).

KCNH2 (hERG1) encodes the alpha-subunit proteins for the rapidly activating delayed rectifier K+ current (I(Kr)), a major K+ current for cardiac myocyte repolarization. In isolated myocytes I(Kr) frequently is small in amplitude or absent, yet KCNH2 channels and I(Kr) are targets for drug block or mutations to cause long QT syndrome. We hypothesized that KCNH2 channels and I(Kr) are uniquely sensitive to enzymatic damage. To test this hypothesis, we studied heterologously expressed K+, Na+, and L-type Ca2+ channels, and in ventricular myocytes I(Kr), slowly activating delayed rectifier K+ current (I(Ks)), and inward rectifier K+ current (I(K1)), by using electrophysiological and biochemical methods. 1) Specific exogenous serine proteases (protease XIV, XXIV, or proteinase K) selectively degraded KCNH2 current (I(KCNH2)) and its mature channel protein without damaging cell integrity and with minimal effects on the other channel currents; 2) immature KCNH2 channel protein remained intact; 3) smaller molecular mass KCNH2 degradation products appeared; 4) protease XXIV selectively abolished I(Kr); and 5) reculturing HEK-293 cells after protease exposure resulted in the gradual recovery of I(KCNH2) and its mature channel protein over several hours. Thus the channel protein for I(KCNH2) and I(Kr) is uniquely sensitive to proteolysis. Analysis of the degradation products suggests selective proteolysis within the S5-pore extracellular linker, which is structurally unique among Kv channels. These data provide 1) a new mechanism to account for low I(Kr) density in some isolated myocytes, 2) evidence that most complexly glycosylated KCNH2 channel protein is in the plasma membrane, and 3) new insight into the rate of biogenesis of KCNH2 channel protein within cells.

Animals↗

Common human SCN5A polymorphisms have altered electrophysiology when expressed in Q1077 splice variants.

BACKGROUND: Eight common (>0.5%) polymorphisms of SCN5A have been described in the US population. Every human also continuously generates two wild-type (WT) splice variants, one with a glutamine residue at position 1077 (Q1077) and one lacking this glutamine (Q1077del). One polymorphism (H558R) has been studied in both splice variants, five polymorphisms (R34C, R481W, S524Y, P1090L,V1951L) have not been previously studied, and two polymorphisms (S1103Y and R1193Q) have been studied in only one of the two splice variants. OBJECTIVES: The purpose of this study was to examine the electrophysiologic molecular phenotype of the eight common polymorphisms in the two human splice variants of SCN5A. METHODS: Currents from 16 channels (all polymorphisms in both splice variants) were determined by voltage clamp and compared with WT after expression in HEK-293 cells. RESULTS: Six of eight polymorphisms showed a distinct phenotype that depended upon the background splice variant used for expression. Only R34C and V1951L showed no functional differences. S524Y showed a dramatic reduction in current density in the Q1077 background similar to that previously described for H558R. Four other polymorphisms (R481W, P1090L, S1103Y, R1193Q) showed shifts in activation, inactivation, or recovery that depended upon the splice variants. Shifts of a similar magnitude have been reported for arrhythmia syndrome mutations and are thought to be pathogenic. CONCLUSION: The majority of common human SCN5A polymorphisms have a distinct molecular phenotype that depends upon the splice variant background. These findings have implications for the interpretation of previous studies of arrhythmia mutations. The significance of these findings for clinical arrhythmia remains to be elucidated.

Alternative Splicing↗

Increased late sodium current in myocytes from a canine heart failure model and from failing human heart.

Electrophysiological remodeling of ion channels in heart failure causes action potential prolongation and plays a role in arrhythmia mechanism. The importance of down-regulation of potassium currents is well-known, but a role for Na current (I(Na)) in heart failure is less well established. We studied I(Na) in heart failure ventricular cells from a canine pacing model of heart failure and also from explanted failing human hearts. Peak I(Na) density was significantly decreased by 39% and 57% in the dog model and in human heart failure, respectively. The kinetics of peak I(Na) were not different in heart failure. Late I(Na) was measured 750 ms after the initial depolarization as the saxitoxin (STX)-sensitive current and also as the current remaining after contaminating currents were blocked. Late I(Na) as a percentage of the peak I(Na) was significantly increased in both conditions. In dogs, STX sensitive late I(Na) was 0.5 +/- 0.1% n = 16 cells from eight normal hearts and 3.4 +/- 1.4% n = 12 cells from seven failing hearts; in humans, it was 0.2 +/- 0.1% n = 4 cells from two normal hearts and 2.4 +/- 0.5% n = 10 cells from three human failing hearts (-40 mV). Quantitative measures of mRNA including RNase protection assays and real time quantitative PCR in the dog model showed no differences for different alpha subunit isoforms (NaV1.1, 1.3, 1.5) and for the beta1 and beta2 subunits. This suggests neither alpha subunit isoform switching nor altered beta subunit expression is a mechanism for increased late I(Na). We conclude that a peak I(Na) is decreased, and non-inactivating late I(Na) is increased in heart failure and this may contribute to action potential prolongation and the generation of arrhythmia.

Animals↗

A trafficking defective, Brugada syndrome-causing SCN5A mutation rescued by drugs.

BACKGROUND: The human cardiac SCN5A gene encodes for the alpha subunit of the human cardiac voltage-dependent sodium channel hNav1.5 [Neuron 28 (2) (2000) 365] and carries inward Na current (INa). Mutations in SCN5A cause arrhythmia syndromes including Brugada syndrome (BrS) and congenital long QT syndrome subtype 3 (LQT3). Here, we report a trafficking defective BrS-causing SCN5A mutation that was drug-rescued. METHODS AND RESULTS: A 14-year-old Caucasian male was diagnosed with BrS with typical ECG pattern for BrS and ventricular fibrillation was easily induced. He also had significant HV interval delay ( approximately 65 ms) and high (31 J) defibrillation thresholds (DFTs). Genomic analysis revealed the SCN5A mutation (G1743R). We engineered G1743R into the cardiac Na channel and transfected HEK-293 cells for functional studies. The mutant channel yielded nearly undetectable sodium channel currents. Coexpression with the beta1 subunit, or incubation at low temperature did not increase current density. However, mexiletine, a sodium channel blocker, increased current density 93-fold in G1743R, but only twofold in WT. CONCLUSIONS: This study identifies an expression-defective BrS mutation in SCN5A with pharmacological rescue. The profoundly decreased sodium current associated with the G1743R suggests a molecular basis for the delayed His-Purkinje conduction and elevated DFTs observed in the proband. Whether the mutant channel may be rescued in vivo by mexiletine and normalize the patient's electrophysiologic parameters remains to be tested.

Adolescent↗

A ubiquitous splice variant and a common polymorphism affect heterologous expression of recombinant human SCN5A heart sodium channels.

Amino acid sequence variations in SCN5A are known to affect function of wild-type channels and also those with coexisting mutations; therefore, it is important to know the exact sequence and function of channels most commonly present in human myocardium. SCN5A was analyzed in control panels of human alleles, demonstrating that the existing clones (hH1, hH1a, hH1b) each contained a rare variant and thus none represented the common sequence. Confirming prior work, the H558R polymorphism was present in approximately 30% of subjects. Quantitative mRNA analysis from human hearts showed that a shorter 2015 amino acid splice variant lacking glutamine at position 1077 (Q1077del) made up 65% of the transcript in every heart examined. Age, sex, race, or structural heart disease did not affect this proportion of Q1077del. Estimated population frequencies for the four common variants were 25% SCN5A, 10% [H558R], 45% [Q1077del], and 20% [H558R;Q1077del], where the reference sequence SCN5A is GenBank AC137587. When expressed in HEK-293 cells, these common variants had a more positive mid-point of the voltage dependence of inactivation than the standard clone hH1. Also, channels containing Q1077 expressed smaller currents. When H558R was present with Q1077 ([H558R]), current expression was profoundly reduced despite normal trafficking to the cell surface. Thus, four variant sequences for SCN5A are commonly present in human myocardium and they exhibit functional differences among themselves and with the previous standard clone. These results have implications for the choice of background sequence for experiments with heterologous expression systems, and possibly implications for electrophysiological function in vivo.

Black or African American↗

A common human SCN5A polymorphism modifies expression of an arrhythmia causing mutation.

SCN5A encodes the alpha-subunit of the ion channel that carries Na current in human heart. From a human cardiac cDNA library we recloned SCN5A. The new clone hH1b differed from existing clones hH1 in four and from hH1a in three positions. The common polymorphism H558R was uniquely present in hH1b. Voltage clamp study showed minor but potentially important kinetic differences between hH1b and the other clones. More dramatically, when the LQT3 mutation M1766L was introduced into the different clones, Na current was markedly reduced in the hH1 and hH1a backgrounds, whereas in hH1b the Na current was not reduced. Immunocytochemistry experiments showed a trafficking defect for M1766L Na channels in hH1 and hH1a but not in hH1b. The double-mutation M1766L/H558R in the hH1a background restored normal trafficking and current including persistent late current, suggesting the disease phenotype was the result of a "double hit" that included the common polymorphism, H558R. These results show that the choice of background clone must be carefully considered in mutagenesis studies. This also represents an example of intragenic complementation, the first for such a large protein.

Amino Acid Substitution↗

A novel SCN5A arrhythmia mutation, M1766L, with expression defect rescued by mexiletine.

OBJECTIVE: Mutations in the cardiac sodium channel gene, SCN5A, cause congenital long QT syndrome (LQT3), Brugada syndrome, idiopathic ventricular fibrillation, and conduction disease by distinct cellular and clinical electrophysiological phenotypes. METHODS: Postmortem molecular analysis of SCN5A was conducted on an infant who presented shortly after birth with self-terminating torsades de pointes. The infant was treated with lidocaine, propranolol, and mexiletine and was stable for 16 months manifesting only a prolonged QT interval. The infant collapsed suddenly following presumed viral gastroenteritis, was found in 2:1 AV block, and was subsequently declared brain dead. Genomic DNA was subjected to SCN5A mutational analyses and DNA sequencing revealing a novel, spontaneous germline missense mutation, M1766L. The M1766L mutation was engineered into the hH1a clone by site-directed mutagenesis, transfected into embryonic kidney cells (HEK-293), and studied by voltage clamp. RESULTS: The M1766L mutation caused a significant decrease in the sodium channel expression. Co-expression with beta1 subunit, incubation at low temperature, and most effectively incubation with mexiletine partially 'rescued' the defective expression. In addition to this pronounced loss of function, M1766L also showed a 10-fold increase in the persistent late sodium current. CONCLUSIONS: These findings suggest that M1766L-SCN5A channel dysfunction may contribute to the basis of lethal arrhythmias, displays an overlapping electrophysiological phenotype, and represents the first sodium channelopathy rescued by drug.

Anti-Arrhythmia Agents↗

Late Na currents affected by alpha subunit isoform and beta1 subunit co-expression in HEK293 cells.

Peak Na current underlies excitability and conduction in the heart, and late non-inactivating or slowly inactivating Na current plays a role in action potential duration. We hypothesized that different alpha subunit isoforms or beta1 subunit co-expression might affect late Na current. The human Na channel alpha subunits hNa(v)1.5 (hH1a) and hNa(v)1.4 (hSkM1) were transfected with and without the hNa(V)beta1 (beta1) subunit in HEK293 cells and studied by whole cell patch clamp. The inactivation relationship for hH1a was 28mV negative to that for hSkM1, and beta1 shifted the midpoint positively by 22mV for hH1a and 8mV for hSkM1. When pre-pulse duration was varied from 10ms to 10s, "steady-state" was approached more slowly for hH1a. beta1 caused hH1a but not hSkM1 to reach "steady-state" earlier. Both isoforms showed two recovery components but hH1a showed a "cardiac phenotype" with a smaller slow component that was unaffected by beta1. The amplitude of a late current (at 750ms) was significantly greater for hH1a than hSkM1, but beta1 decreased late current for hH1a and eliminated the difference. Under the study conditions the alpha subunit isoforms have distinct functional phenotypes and co-expression with beta1 tends to diminish these distinctions. These properties may provide mechanisms for regional and transmural distribution of late Na current and late Na current amplitudes during development and in disease states.

Humans↗