Persistent PKA activation redistributes NaV1.5 to the cell surface of adult rat ventricular myocytes.

During chronic stress, persistent activation of cAMP-dependent protein kinase (PKA) occurs, which can contribute to protective or maladaptive changes in the heart. We sought to understand the effect of persistent PKA activation on NaV1.5 channel distribution and function in cardiomyocytes using adult rat ventricular myocytes as the main model. PKA activation with 8CPT-cAMP and okadaic acid (phosphatase inhibitor) caused an increase in Na+ current amplitude without altering the total NaV1.5 protein level, suggesting a redistribution of NaV1.5 to the myocytes' surface. Biotinylation experiments in HEK293 cells showed that inhibiting protein trafficking from intracellular compartments to the plasma membrane prevented the PKA-induced increase in cell surface NaV1.5. Additionally, PKA activation induced a time-dependent increase in microtubule plus-end binding protein 1 (EB1) and clustering of EB1 at myocytes' peripheral surface and intercalated discs (ICDs). This was accompanied by a decrease in stable interfibrillar microtubules but an increase in dynamic microtubules along the myocyte surface. Imaging and coimmunoprecipitation experiments revealed that NaV1.5 interacted with EB1 and ß-tubulin, and both interactions were enhanced by PKA activation. We propose that persistent PKA activation promotes NaV1.5 trafficking to the peripheral surface of myocytes and ICDs by providing dynamic microtubule tracks and enhanced guidance by EB1. Our proposal is consistent with an increase in the correlative distribution of NaV1.5, EB1, and ß-tubulin at these subcellular domains in PKA-activated myocytes. Our study suggests that persistent PKA activation, at least during the initial phase, can protect impulse propagation in a chronically stressed heart by increasing NaV1.5 at ICDs.

Dual targeting of MTOR as a novel therapeutic approach for high-risk B-cell acute lymphoblastic leukemia.

Children of Hispanic/Latino ancestry have increased incidence of high-risk B-cell acute lymphoblastic leukemia (HR B-ALL) with poor prognosis. This leukemia is characterized by a single-copy deletion of the IKZF1 (IKAROS) tumor suppressor and increased activation of the PI3K/AKT/mTOR pathway. This identifies mTOR as an attractive therapeutic target in HR B-ALL. Here, we report that IKAROS represses MTOR transcription and IKAROS' ability to repress MTOR in leukemia is impaired by oncogenic CK2 kinase. Treatment with the CK2 inhibitor, CX-4945, enhances IKAROS activity as a repressor of MTOR, resulting in reduced expression of MTOR in HR B-ALL. Thus, we designed a novel therapeutic approach that implements dual targeting of mTOR: direct inhibition of the mTOR protein (with rapamycin), in combination with IKAROS-mediated transcriptional repression of the MTOR gene (using the CK2 inhibitor, CX-4945). Combination treatment with rapamycin and CX-4945 shows synergistic therapeutic effects in vitro and in patient-derived xenografts from Hispanic/Latino children with HR B-ALL. These data suggest that such therapy has the potential to reduce the health disparity in HR B-ALL among Hispanic/Latino children. The dual targeting of oncogene transcription, combined with inhibition of the corresponding oncoprotein provides a paradigm for a novel precision medicine approach for treating hematological malignancies.

Expression defect of the rare variant/Brugada mutation R1512W depends upon the SCN5A splice variant background and can be rescued by mexiletine and the common polymorphism H558R.

Background : Mutations in SCN5A that decrease Na current underlie arrhythmia syndromes such as the Brugada syndrome (BrS). 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 R1512W, a mutation reported to cause BrS. Methods and results : We made the mutation in both variants and expressed them in HEK-293 cells for voltage-clamp study. After 24 hours of transfection, the current expression level of R1512W was reduced by ~50% in both Q1077del and Q1077 compared to the wild-type (WT) channel, respectively. The activation and inactivation midpoint were not different between WT and mutant channels in both splice variant backgrounds. However, slower time constants of recovery and enhanced intermediate inactivation were observed for R1512W/Q1077 compared with WT-Q1077, while the recovery and intermediate inactivation parameters of R1512W/Q1077del were similar to WT-Q1077del. Furthermore, both mexiletine and the common polymorphism H558R restored peak sodium current (INa) amplitude of the mutant channel by increasing the cell surface expression of SCN5A. Conclusion : These findings provide further evidence that the splice variant affects the molecular phenotype with implications for the clinical phenotype, and they provide insight into the expression defect mechanisms and potential treatment in BrS.

A novel G6PD deleterious variant identified in three families with severe glucose-6-phosphate dehydrogenase deficiency.

BACKGROUND: Glucose-6-phosphate dehydrogenase deficiency (D-G6PD) is an X-linked recessive disorder resulted from deleterious variants in the housekeeping gene Glucose-6-phosphate 1-dehydrogenase (G6PD), causing impaired response to oxidizing agents. Screening for new variations of the gene helps with early diagnosis of D-G6PD resulting in a reduction of disease related complications and ultimately increased life expectancy of the patients. METHODS: One thousand five hundred sixty-five infants with pathological jaundice were screened for G6PD variants by Sanger sequencing all of the 13 exons, and the junctions of exons and introns of the G6PD gene. RESULTS: We detected G6PD variants in 439 (28.1%) of the 1565 infants with pathological jaundice. In total, 9 types of G6PD variants were identified in our cohort; and a novel G6PD missense variant c.1118 T > C, p.Phe373Ser in exon 9 of the G6PD gene was detected in three families. Infants with this novel variant showed decreased activity of G6PD, severe anemia, and pathological jaundice, consistent with Class I G6PD deleterious variants. Analysis of the resulting protein's structure revealed this novel variant affects G6PD protein stability, which could be responsible for the pathogenesis of D-G6PD in these patients. CONCLUSIONS: High rates of G6PD variants were detected in infants with pathological jaundice, and a novel Class I G6PD deleterious variants was identified in our cohort. Our data reveal that variant analysis is helpful for the diagnosis of D-G6PD in patients, and also for the expansion of the spectrum of known G6PD variants used for carrier detection and prenatal diagnosis.

IKAROS and CK2 regulate expression of BCL-XL and chemosensitivity in high-risk B-cell acute lymphoblastic leukemia.

High-risk B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive disease, often characterized by resistance to chemotherapy. A frequent feature of high-risk B-ALL is loss of function of the IKAROS (encoded by the IKZF1 gene) tumor suppressor. Here, we report that IKAROS regulates expression of the BCL2L1 gene (encodes the BCL-XL protein) in human B-ALL. Gain-of-function and loss-of-function experiments demonstrate that IKAROS binds to the BCL2L1 promoter, recruits histone deacetylase HDAC1, and represses BCL2L1 expression via chromatin remodeling. In leukemia, IKAROS' function is impaired by oncogenic casein kinase II (CK2), which is overexpressed in B-ALL. Phosphorylation by CK2 reduces IKAROS binding and recruitment of HDAC1 to the BCL2L1 promoter. This results in a loss of IKAROS-mediated repression of BCL2L1 and increased expression of BCL-XL. Increased expression of BCL-XL and/or CK2, as well as reduced IKAROS expression, are associated with resistance to doxorubicin treatment. Molecular and pharmacological inhibition of CK2 with a specific inhibitor CX-4945, increases binding of IKAROS to the BCL2L1 promoter and enhances IKAROS-mediated repression of BCL2L1 in B-ALL. Treatment with CX-4945 increases sensitivity to doxorubicin in B-ALL, and reverses resistance to doxorubicin in multidrug-resistant B-ALL. Combination treatment with CX-4945 and doxorubicin show synergistic therapeutic effects in vitro and in preclinical models of high-risk B-ALL. Results reveal a novel signaling network that regulates chemoresistance in leukemia. These data lay the groundwork for clinical testing of a rationally designed, targeted therapy that combines the CK2 inhibitor, CX-4945, with doxorubicin for the treatment of hematopoietic malignancies.

Two novel deleterious variants of Angiotensin-I-converting Enzyme gene identified in a family with recurrent anhydramnios.

BACKGROUND: Anhydramnios results from the poor development of the placenta or problems with intrauterine development of the kidneys or urinary tract. Complete lack of amniotic fluid indicates a severe problem with the organs of the urinary system. The genes associated with anhydramnios show very diversity and are not yet well defined. METHODS: Whole-exome sequencing (WES) was used for an aborted male case around the 20th week of gestation diagnosed with anhydramnios. The resulted deleterious variants were verified by Sanger sequencing. Pathogenicity of deleterious variants was explored by in silico analysis. RESULTS: A maternally inherited deleterious frameshift variant, c.1454_1455insC, p.(S486Ffs29) in exon 9 and two paternally inherited missense variants c.1037C > G, p.(Ser346Trp) in exon 7 and c.1465A > G, p.(Asn489Asp) in exon 9 of Angiotensin-I-Converting Enzyme (ACE) gene were found and confirmed by Sanger sequencing. c.1454_1455insC, p.(S486Ffs29) and c.1037C > G, p.(Ser346Trp) were identified as two novel compound heterozygous deleterious variants. The pathogenicity of these deleterious variants was determined by in silico analysis and both the deleterious variants disrupt the structure of the ACE protein. CONCLUSION: Two novel compound heterozygous variants were identified in the case with anhydramnios, which may be associated with pathogenicity of anhydramnios. Our data also revealed that the WES approach may provide helpful information for genetic counseling of the families with anhydramnios.

Mexiletine rescues a mixed biophysical phenotype of the cardiac sodium channel arising from the SCN5A mutation, N406K, found in LQT3 patients.

INTRODUCTION: Individual mutations in the SCN5A-encoding cardiac sodium channel α-subunit usually cause a single cardiac arrhythmia disorder, some cause mixed biophysical or clinical phenotypes. Here we report an infant, female patient harboring a N406K mutation in SCN5A with a marked and mixed biophysical phenotype and assess pathogenic mechanisms. METHODS AND RESULTS: A patient suffered from recurrent seizures during sleep and torsades de pointes with a QTc of 530 ms. Mutational analysis identified a N406K mutation in SCN5A. The mutation was engineered by site-directed mutagenesis and heterologously expressed in HEK293 cells. After 48 hours incubation with and without mexiletine, macroscopic voltage-gated sodium current (INa) was measured with standard whole-cell patch clamp techniques. SCN5A-N406K elicited both a significantly decreased peak INa and a significantly increased late INa compared to wide-type (WT) channels. Furthermore, mexiletine both restored the decreased peak INa of the mutant channel and inhibited the increased late INa of the mutant channel. CONCLUSION: SCN5A-N406K channel displays both "gain-of-function" in late INa and "loss-of-function" in peak INa density contributing to a mixed biophysical phenotype. Moreover, our finding may provide the first example that mexiletine exerts a dual rescue of both "gain-of-function" and "loss-of-function" of the mutant sodium channel.

Casein Kinase II (CK2) as a Therapeutic Target for Hematological Malignancies.

BACKGROUND: Casein kinase II (CK2) is a pro-oncogenic protein, which is emerging as a promising therapeutic target in cancer. Recent studies have revealed an important role for CK2 in tumorigenesis. High levels of CK2 are noted in many malignancies including leukemia. Use of CK2 inhibitors in various malignancies including breast, prostate, and lung cancer are being tested. Although many CK2 inhibitors exist, only a few have emerged as selective inhibitors that are potent and effective. CX-4945 is a selective, orallybioavailable small molecule inhibitor, which has shown encouraging results in pre-clinical models of leukemia. METHODS: In this review we will elaborate on the structure and physiological function of the CK2 protein as well as its role in cancer. We will review, in depth, the role of CK2 in leukemia and its mechanisms of tumorigenesis via phosphorylation of the tumor suppressor protein Ikaros. We will discuss both the importance of Ikaros in leukemia suppression and the restoration of Ikaros' tumor suppressor function after CK2 inhibition by CX-4945 (a CK2-specific inhibitor). RESULTS: CK2 is an oncogene that is overexpressed in hematological malignancies. In high risk Pre-B ALL, CK2 phosphorylates Ikaros tumor suppressor and promotes leukemogenesis. Inhibition of CK2 using CX4945 restores Ikaros function and leads to anti leukemic effects in vitro and in pre-clinical leukemia models. CONCLUSION: CK2 is an attractive target in treatment of various cancers. Currently only a few specific CK2 inhibitors are available. Preclinical studies using CK2 inhibitor, CX4945 in high risk pediatric leukemias have shown promising results and warrants further testing in other types of leukemia.

Transcriptional Regulation of JARID1B/KDM5B Histone Demethylase by Ikaros, Histone Deacetylase 1 (HDAC1), and Casein Kinase 2 (CK2) in B-cell Acute Lymphoblastic Leukemia.

Impaired function of the Ikaros (IKZF1) protein is associated with the development of high-risk B-cell precursor acute lymphoblastic leukemia (B-ALL). The mechanisms of Ikaros tumor suppressor activity in leukemia are unknown. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. Here, we report that Ikaros represses transcription of the histone H3K4 demethylase, JARID1B (KDM5B). Transcriptional repression of JARID1B is associated with increased global levels of H3K4 trimethylation. Ikaros-mediated repression of JARID1B is dependent on the activity of the histone deacetylase, HDAC1, which binds to the upstream regulatory element of JARID1B in complex with Ikaros. In leukemia, JARID1B is overexpressed, and its inhibition results in cellular growth arrest. Ikaros-mediated repression of JARID1B in leukemia is impaired by pro-oncogenic casein kinase 2 (CK2). Inhibition of CK2 results in increased binding of the Ikaros-HDAC1 complex to the promoter of JARID1B, with increased formation of trimethylated histone H3 lysine 27 and decreased histone H3 Lys-9 acetylation. In cases of high-risk B-ALL that carry deletion of one Ikaros (IKZF1) allele, targeted inhibition of CK2 restores Ikaros binding to the JARID1B promoter and repression of JARID1B. In summary, the presented data suggest a mechanism through which Ikaros and HDAC1 regulate the epigenetic signature in leukemia: via regulation of JARID1B transcription. The presented data identify JARID1B as a novel therapeutic target in B-ALL and provide a rationale for the use of CK2 inhibitors in the treatment of high-risk B-ALL.

MiR-215, an activator of the CTNNBIP1/ß-catenin pathway, is a marker of poor prognosis in human glioma.

MicroRNA-215 (miR-215) promotes tumor growth in various human malignancies. However, its role has not yet been determined in human glioma. Here, we found that levels of miR-215 were higher in glioma tissues than in corresponding non-neoplastic brain tissue. High miR-215 expression was correlated with higher World Health Organization (WHO) grades and shorter overall survival. Multivariate and univariate analysis indicated that miR-215 expression was an independent prognostic factor. We also found that TGF-beta1, phosphorylated beta-catenin, alpha-SMA, and fibronectin were increased in glioma tissues. Additionally, CTNNBIP1, a direct target of miR-215, was decreased in glioma compared to adjacent normal tissue. These data indicate that miR-215 activates Wnt/ß-catenin signaling by increasing ß-catenin phosphorylation, α-SMA expression, and fibronectin expression. It promotes TGF-ß1-induced oncogenesis by suppressing CTNNBIP1 in glioma. In summary, miR-215 is overexpressed in human glioma, is involved in TGF-ß1-induced oncogenesis, and can be used as a marker of poor prognosis in glioma patients.

Targeting casein kinase II restores Ikaros tumor suppressor activity and demonstrates therapeutic efficacy in high-risk leukemia.

Ikaros (IKZF1) is a tumor suppressor that binds DNA and regulates expression of its target genes. The mechanism of Ikaros activity as a tumor suppressor and the regulation of Ikaros function in leukemia are unknown. Here, we demonstrate that Ikaros controls cellular proliferation by repressing expression of genes that promote cell cycle progression and the phosphatidylinositol-3 kinase (PI3K) pathway. We show that Ikaros function is impaired by the pro-oncogenic casein kinase II (CK2), and that CK2 is overexpressed in leukemia. CK2 inhibition restores Ikaros function as transcriptional repressor of cell cycle and PI3K pathway genes, resulting in an antileukemia effect. In high-risk leukemia where one IKZF1 allele has been deleted, CK2 inhibition restores the transcriptional repressor function of the remaining wild-type IKZF1 allele. CK2 inhibition demonstrated a potent therapeutic effect in a panel of patient-derived primary high-risk B-cell acute lymphoblastic leukemia xenografts as indicated by prolonged survival and a reduction of leukemia burden. We demonstrate the efficacy of a novel therapeutic approach for high-risk leukemia: restoration of Ikaros tumor suppressor activity via inhibition of CK2. These results provide a rationale for the use of CK2 inhibitors in clinical trials for high-risk leukemia, including cases with deletion of one IKZF1 allele.

Arrhythmogenic Biophysical Phenotype for SCN5A Mutation S1787N Depends upon Splice Variant Background and Intracellular Acidosis.

BACKGROUND: SCN5A is a susceptibility gene for type 3 long QT syndrome, Brugada syndrome, and sudden infant death syndrome. INa dysfunction from mutated SCN5A can depend upon the splice variant background in which it is expressed and also upon environmental factors such as acidosis. S1787N was reported previously as a LQT3-associated mutation and has also been observed in 1 of 295 healthy white controls. Here, we determined the in vitro biophysical phenotype of SCN5A-S1787N in an effort to further assess its possible pathogenicity. METHODS AND RESULTS: We engineered S1787N in the two most common alternatively spliced SCN5A isoforms, the major isoform lacking a glutamine at position 1077 (Q1077del) and the minor isoform containing Q1077, and expressed these two engineered constructs in HEK293 cells for electrophysiological study. Macroscopic voltage-gated INa was measured 24 hours after transfection with standard whole-cell patch clamp techniques. We applied intracellular solutions with pH7.4 or pH6.7. S1787N in the Q1077 background had WT-like INa including peak INa density, activation and inactivation parameters, and late INa amplitude in both pH 7.4 and pH 6.7. However, with S1787N in the Q1077del background, the percentages of INa late/peak were increased by 2.1 fold in pH 7.4 and by 2.9 fold in pH 6.7 when compared to WT. CONCLUSION: The LQT3-like biophysical phenotype for S1787N depends on both the SCN5A splice variant and on the intracellular pH. These findings provide further evidence that the splice variant and environmental factors affect the molecular phenotype of cardiac SCN5A-encoded sodium channel (Nav1.5), has implications for the clinical phenotype, and may provide insight into acidosis-induced arrhythmia mechanisms.

The interaction of caveolin 3 protein with the potassium inward rectifier channel Kir2.1: physiology and pathology related to long qt syndrome 9 (LQT9).

Mutations in CAV3 cause LQT syndrome 9 (LQT9). A previously reported LQT9 patient had prominent U waves on ECG, a feature that has been correlated with Kir2.1 loss of function. Our objective was to determine whether caveolin 3 (Cav3) associates with Kir2.1 and whether LQT9-associated CAV3 mutations affect the biophysical properties of Kir2.1. Kir2.1 current (IK1) density was measured using the whole-cell voltage clamp technique. WT-Cav3 did not affect IK1. However, F97C-Cav3 and T78M-Cav3 decreased IK1 density significantly by ∼60%, and P104L-Cav3 decreased IK1 density significantly by ∼30% at -60 mV. Immunostained rat heart cryosections and HEK293 cells cotransfected with Kir2.1 and WT-Cav3 both demonstrated colocalization of Kir2.1 and WT-Cav3 by confocal imaging. Cav3 coimmunoprecipitated with Kir2.1 in human ventricular myocytes and in heterologous expression systems. Additionally, FRET efficiency was highly specific, with a molecular distance of 5.6 ± 0.4 nm, indicating close protein location. Colocalization experiments found that Cav3 and Kir2.1 accumulated in the Golgi compartment. On-cell Western blot analysis showed decreased Kir2.1 cell surface expression by 60% when expressed with F97C-Cav3 and by 20% when expressed with P104L-Cav3 compared with WT-Cav3. This is the first report of an association between Cav3 and Kir2.1. The Cav3 mutations F97C-Cav3, P104L-Cav3, and T78M-Cav3 decreased IK1 density significantly. This effect was related to a reduced cell surface expression of Kir2.1. Kir2.1 loss of function is additive to the increase described previously in late INa, prolonging repolarization and leading to arrhythmia generation in Cav3-mediated LQT9.

Digenic inheritance novel mutations in SCN5a and SNTA1 increase late I(Na) contributing to LQT syndrome.

SCN5A and SNTA1 are reported susceptible genes for long QT syndrome (LQTS). This study was designed to elucidate a plausible pathogenic arrhythmia mechanism for the combined novel mutations R800L-SCN5A and A261V-SNTA1 on cardiac sodium channels. A Caucasian family with syncope and marginally prolonged QT interval was screened for LQTS-susceptibility genes and found to harbor the R800L mutation in SCN5A and A261V mutation in SNTA1, and those with both mutations had the strongest clinical phenotype. The mutations were engineered into the most common splice variant of human SCN5A and SNTA1 cDNA, respectively, and sodium current (INa) was characterized in human embryonic kidney 293 cells cotransfected with neuronal nitric oxide synthase (nNOS) and the cardiac isoform of the plasma membrane Ca-ATPase (PMCA4b). Peak INa densities were unchanged for wild-type (WT) and for mutant channels containing R800L-SCN5A, A261V-SNTA1, or R800L-SCN5A plus A261V-SNTA1. However, late INa for either single mutant was moderately increased two- to threefold compared with WT. The combined mutations of R800L-SCN5A plus A261V-SNTA1 significantly enhanced the INa late/peak ratio by 5.6-fold compared with WT. The time constants of current decay of combined mutant channel were markedly increased. The gain-of-function effect could be blocked by the N(G)-monomethyl-l-arginine, a nNOS inhibitor. We conclude that novel mutations in SCN5A and SNTA1 jointly exert a nNOS-dependent gain-of-function on SCN5A channels, which may consequently prolong the action potential duration and lead to LQTS phenotype.

Loss-of-function mutations in the KCNJ8-encoded Kir6.1 K(ATP) channel and sudden infant death syndrome.

BACKGROUND: Approximately 10% of sudden infant death syndrome (SIDS) may stem from cardiac channelopathies. The KCNJ8-encoded Kir6.1 (K(ATP)) channel critically regulates vascular tone and cardiac adaptive response to systemic metabolic stressors, including sepsis. KCNJ8-deficient mice are prone to premature sudden death, particularly with infection. We determined the spectrum, prevalence, and function of KCNJ8 mutations in a large SIDS cohort. METHODS AND RESULTS: Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing, comprehensive open reading frame/splice-site mutational analysis of KCNJ8 was performed on genomic DNA isolated from necropsy tissue on 292 unrelated SIDS cases (178 males, 204 white; age, 2.9±1.9 months). KCNJ8 mutations were coexpressed heterologously with SUR2A in COS-1 cells and characterized using whole-cell patch-clamp. Two novel KCNJ8 mutations were identified. A 5-month-old white male had an in-frame deletion (E332del) and a 2-month-old black female had a missense mutation (V346I). Both mutations localized to Kir6.1's C-terminus, involved conserved residues and were absent in 400 and 200 ethnic-matched reference alleles respectively. Both cases were negative for mutations in established channelopathic genes. Compared with WT, the pinacidil-activated K(ATP) current was decreased 45% to 68% for Kir6.1-E332del and 40% to 57% for V346I between -20 mV and 40 mV. CONCLUSIONS: Molecular and functional evidence implicated loss-of-function KCNJ8 mutations as a novel pathogenic mechanism in SIDS, possibly by predisposition of a maladaptive cardiac response to systemic metabolic stressors akin to the mouse models of KCNJ8 deficiency.

The common African American polymorphism SCN5A-S1103Y interacts with mutation SCN5A-R680H to increase late Na current.

The common polymorphism SCN5A-S1103Y (∼13% allelic frequency in African Americans) is a risk factor for arrhythmia, sudden unexplained death (SUD), and sudden infant death syndrome. Prompted by a case of autopsy-negative SUD in a 23-year-old African American man who collapsed while playing football, we hypothesized that S1103Y interacted with other SCN5A variants to pathologically modify sodium current (I(Na)). Mutational analysis of arrhythmia-associated genes in the victim revealed the variants SCN5A-R680H and SCN5A-S1103Y. These variants were made both separately and in the same cDNA construct of the alternative splice variant backgrounds (SCN5A-Q1077del and Q1077) and expressed in HEK293 cells. In the most abundant SCN5A-Q1077del, late I(Na) for S1103Y alone was not significantly different from wild type (WT). However, late I(Na) for R680H, R680H+S1103Y (coexpressed), and R680H/S1103Y (on the same cDNA) was increased 2.1-, 3.4-, and 3.6-fold, respectively, compared with WT. Intracellular acidosis (pH 6.7) increased late I(Na) for S1103Y, R680H, R680H+S1103Y, and R680H/S1103Y by 2.2-, 2.4-, 5.0-, and 5.5-fold, respectively, compared with WT at pH 6.7. Expression in the less abundant SCN5A-Q1077 showed no increased late I(Na). This is the initial report of a functional interaction for the common polymorphism S1103Y with another mutation in the major transcript Q1077del of SCN5A. The "double hit" and environmental factor of acidosis may have converged to cause arrhythmic sudden death in this case.

Sudden unexplained nocturnal death syndrome in Southern China: an epidemiological survey and SCN5A gene screening.

Based on autopsy data collected in Southern China from 2001-2006, 975 cases of sudden unexplained nocturnal death syndrome (SUNDS) were surveyed. Genetic screening of SCN5A gene encoding the voltage dependent cardiac sodium channel was performed in 74 SUNDS cases. The annual occurrence rate of SUNDS in the area was estimated to be about 1 per 100,000 people. About 80.6% of deaths occurred between the ages of 21 to 40 years and the case number peaked at age 30 years. In 75.4% of cases with witnesses, victims died asleep between 11 PM and 4 AM and they showed predominantly abrupt respiratory distress shortly preceding death. The monthly distribution of emergency fever cases in the area during the same period was positively correlated to that of SUNDS cases (r(s) = 0.611, P = 0.035). Four polymorphisms in SCN5A were identified in both SUNDS and control groups. Compared with controls, the allele frequency of C5457 and C3666 + 69 were significant higher in SUNDS (P < 0.005) while the genotypes of both 5457CC (P = 0.012, OR = 2.0, 95% CI = 1.3-3.2) and 3666+69CC (P = 0.004, OR = 2.1, 95% CI = 1.3-3.3) in SUNDS cases were significantly higher. This is the first report of an epidemiological survey and SCN5A gene screening in SUNDS in the Han population of China. The genotypes of 5457CC and 3666+69CC in SCN5A gene may be Chinese SUNDS susceptible polymorphisms.

LQTS-associated mutation A257G in α1-syntrophin interacts with the intragenic variant P74L to modify its biophysical phenotype.

The SNTA1-encoded α1-syntrophin (SNTA1) missense mutation, p.A257G, causes long QT syndrome (LQTS) by pathogenic accentuation of Nav1.5's sodium current (INa). Subsequently, we found p.A257G in combination with the SNTA1 polymorphism, p.P74L in 4 victims of sudden infant death syndrome (SIDS) as well as in 3 adult controls. We hypothesized that p.P74L-SNTA1 could functionally modify the pathogenic phenotype of p.A257G-SNTA1, thus explaining its occurrence in non-LQTS populations. The SNTA1 variants p.P74L, p.A257G, and the combination variant p.P74L/p.A257G were engineered using PCR-based overlap-extension and were co-expressed heterologously with SCN5A in HEK293 cells. INa was recorded using the whole-cell method. Compared to wild-type (WT), the significant increase in peak INa and window current found with p.A257G was reversed by the intragenic variant p.P74L (p.P74L/p.A257G). These results report for the first time the intragenic rescue of an LQT-associated SNTA1 mutation when found in combination with the SNTA1 polymorphism p.P74L, suggesting an ever-increasing picture of complexity in terms of genetic risk stratification for arrhythmia.

Cardiomyocyte sulfonylurea receptor 2-KATP channel mediates cardioprotection and ST segment elevation.

Sulfonylurea receptor-containing ATP-sensitive potassium (K(ATP)) channels have been implicated in cardioprotection, but the cell type and constitution of channels responsible for this protection have not been clear. Mice deleted for the first nucleotide binding region of sulfonylurea receptor 2 (SUR2) are referred to as SUR2 null since they lack full-length SUR2 and glibenclamide-responsive K(ATP) channels in cardiac, skeletal, and smooth muscle. As previously reported, SUR2 null mice develop electrocardiographic changes of ST segment elevation that were shown to correlate with coronary artery vasospasm. Here we restored expression of the cardiomyocyte SUR2-K(ATP) channel in SUR2 null mice by generating transgenic mice with ventricular cardiomyocyte-restricted expression of SUR2A. Introduction of the cardiomyocyte SUR2A transgene into the SUR2 null background restored functional cardiac K(ATP) channels. Hearts isolated from rescued mice, referred to as MLC2A, had significantly reduced infarct size (27 ± 3% of area at risk) compared with SUR2 null mice (36 ± 3% of area at risk). Compared with SUR2 null hearts, MLC2A hearts exhibited significantly improved cardiac function during the postischemia reperfusion period primarily because of preservation of low diastolic pressures. Additionally, restoration of cardiac SUR2-K(ATP) channels significantly reduced the degree and frequency of ST segment elevation episodes in MLC2A mice. Therefore, cardioprotective mechanisms both dependent and independent of SUR2-K(ATP) channels contribute to cardiac function.

Gain-of-function mutation S422L in the KCNJ8-encoded cardiac K(ATP) channel Kir6.1 as a pathogenic substrate for J-wave syndromes.

BACKGROUND: J-wave syndromes have emerged conceptually to encompass the pleiotropic expression of J-point abnormalities including Brugada syndrome (BrS) and early repolarization syndrome (ERS). KCNJ8, which encodes the cardiac K(ATP) Kir6.1 channel, recently has been implicated in ERS following identification of the functionally uncharacterized missense mutation S422L. OBJECTIVE: The purpose of this study was to further explore KCNJ8 as a novel susceptibility gene for J-wave syndromes. METHODS: Using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing, comprehensive open reading frame/splice site mutational analysis of KCNJ8 was performed in 101 unrelated patients with J-wave syndromes, including 87 with BrS and 14 with ERS. Six hundred healthy individuals were examined to assess the allelic frequency for all variants detected. KCNJ8 mutation(s) was engineered by site-directed mutagenesis and coexpressed heterologously with SUR2A in COS-1 cells. Ion currents were recorded using whole-cell configuration of the patch-clamp technique. RESULTS: One BrS case and one ERS case hosted the identical missense mutation S422L, which was reported previously. KCNJ8-S422L involves a highly conserved residue and was absent in 1,200 reference alleles. Both cases were negative for mutations in all known BrS and ERS susceptibility genes. K(ATP) current of the Kir6.1-S422L mutation was increased significantly over the voltage range from 0 to 40 mV compared to Kir6.1-WT channels (n = 16-21; P <.05). CONCLUSION: These findings further implicate KCNJ8 as a novel J-wave syndrome susceptibility gene and a marked gain of function in the cardiac K(ATP) Kir6.1 channel secondary to KCNJ8-S422L as a novel pathogenic mechanism for the phenotypic expression of both BrS and ERS.