- Email: [email protected]
KCNH2 MUTATIONS THAT ALTER KV11.1 CHANNEL GATING ARE LINKED TO SUDDEN INFANT DEATH SYNDROME
2132 tomic tracks of reentrant drivers may be critical points for targeted AF ablation.
LONG QT SYNDROME-ASSOCIATED CAVEOLIN-3 MUTATION F97C IMPARTS A LOSS-OF-FUNCTION EFFECT ON CARDIAC TRANSIENT OUTWARD POTASSIUM CURRENT (ITO) L. Tyan, W.M. Mesquitta, A.C. Grimes, R.C. Balijepalli, University of Wisconsin–Madison, Madison, WI. Background: Caveolae are discrete microdomains in cardiomyocytes that localize many ion channels and signaling proteins. Mutations in the CAV3 gene encoding Caveolin-3 (Cav3), a muscle-specific scaffolding protein integral to caveolae in the cardiomyocytes, have been associated with a congenital long QT syndrome (LQT9). The transient outward potassium current (Ito) is responsible for the rapid repolarization phase of the cardiac action potential (AP) and is one of the major determinants of the length of the QT interval. Kv4.2 and Kv4.3 channels generate the fast (Ito,f) and Kv1.4 channel mediates the slow (Ito,s) components of Ito. Previously we showed that LQTS-associated Cav3 mutations, F97C and S141R, prolong AP duration by increasing sodium channel late current, but whether these mutations impact the Ito is unknown. Methods: We determined the impact of LQTS-associated Cav3 mutations on the function of Kv4.2 and Kv4.3 channels using whole-cell patch-clamp analysis in HEK293 cells, by transiently co-expressing either wild-type (WT) Cav3, the LQT9-associated Cav3 mutations F97C or S141R, or a dominant-negative Cav3 mutation P104L. Co-immunoprecipitation and western blot analysis were performed to determine associations between Kv4.2 and/or Kv4.3 channels and Cav3. Results: Co-expression of F97C or P104 significantly reduced the peak Ikv4.2 and Ikv4.3 densities compared to cells transfected with either WT Cav-3 or vector controls. In contrast, co-expression of S141R did not significantly alter either Ikv4.2 or Ikv4.3. Analysis of activation and inactivation time constants of Kv4.2 and Kv4.3 channels revealed that F97C or P104L caused slower activation and slower inactivation of Ikv4.2 and slower activation of Ikv4.3. The inactivation time constant for Ikv4.3 was not affected. Furthermore, co-immunoprecipitation and western blot analysis revealed that Cav3 associates with Kv4.2 and Kv4.3 channels in mouse ventricular myocytes and transiently expressed HEK293 cells. Conclusions: Cav3 associates with Kv channels that form the Ito,f in cardiomyocytes. Furthermore, the LQT Cav-3 mutation F97C shows important loss-of-function effects on Ito, which will prolong repolarization and may contribute to arrhythmogenesis.
MICE THAT OVEREXPRESS A FRAMESHIFT MUTATION IN HUMAN NATRIURETIC PEPTIDE PRECURSOR A GENE EXHIBIT INCREASED ATRIAL FIBRILLATION BURDEN BUT NO ATRIAL STRUCTURAL REMODELING. E. Savio Galimberti, P. Kannankeril, K. Kor, S. Kupershmidt, S. Ansani, M. Blair, D. Darbar Vanderbilt University, Nashville, TN.
Heart Rhythm, Vol 11, No 11, November 2014 Background: A frameshift mutation in natriuretic peptide precursor A gene (NPPA) encoding a mutant atrial natriuretic peptide (ANP) has been linked with familial atrial fibrillation (AF). Mutant ANP shortened monophasic action potential duration and effective refractory period in retrogradely perfused rat hearts suggesting a potential mechanism for AF. We engineered 2 transgenic mouse lines that overexpress either mutant human NPPA (h-M) or human wild-type NPPA (h-WT) to test the hypothesis that h-M mice are more prone to develop AF. Methods: We determined circulating ANP levels and blood pressure (BP) in h-M, h-WT, and nontransgenic (NTG) B6D2 mice. To assess AF inducibility and burden, we conducted transesophageal pacing studies in anesthetized mice and isoproterenol challenge (ISO) in telemetry-implanted mice. To estimate atrial volumes and assess ventricular function we performed echocardiograms. Finally, we assessed atrial fibrosis in histologic slides dyed with Masson trichrome blue. Results: Circulating h-M-ANP level was 5-fold higher compared to h-WTANP and NTG-ANP (12.0 ⫾ 2.0 vs 4.0 ⫾ 1.0 vs 2.0 ⫾ 0.4 pg/mL, Po.01, n ¼ 5 mice/group), with cardiac tissue levels 6 orders of magnitude higher than blood levels. Mean BP was lower in h-M mice compared to hWT and NTG (68.8 ⫾ 1.0 vs 76.5 ⫾ 2.5 vs 85.1 ⫾ 1.6 mm Hg, Po.01, n ¼ 30 mice/group). h-M mice showed significantly increased AF burden/ animal than either h-WT or NTG (57.0 ⫾ 2.0 vs 5 ⫾ 1.0 vs 11.0 ⫾ 1.0 s, Po.05, n ¼ 5 mice/group). Also, 67% of h-M mice developed AF after ISO, which was more sustained than in NTG (21.3 ⫾ 3.0 vs 1.8 ⫾ 0.4 min, Po.01, n ¼ 3 mice/group), and h-WT mice did not develop AF. There were no changes in atrial volumes or fibrosis across groups. Cardiac output estimated by echo was preserved across groups (h-M: 28 ⫾ 2.0 vs h-WT: 32.2 ⫾ 1.4 vs NTG: 30.7 ⫾ 3.2 mL/min, n ¼ 5 mice/group). Although ejection fraction was similar in h-M (81.8 ⫾ 0.4%) and NTG (80.1 ⫾ 0.9%) and slightly decreased in h-WT (75.2 ⫾ 1.0 %, Po.01, n ¼ 5/group), all 3 groups were within normal range. Conclusions: Overexpression of human NPPA frameshift mutation in mice is associated with increased AF inducibility and burden. Absence of atrial fibrosis and preservation of ventricular function rule out global cardiomyopathy.
KCNH2 MUTATIONS THAT ALTER KV11.1 CHANNEL GATING ARE LINKED TO SUDDEN INFANT DEATH SYNDROME J.L. Smith,1 D.J. Tester,2 A.R. Reloj,1 D.E. Burgess,1 C. Hsu,1 M.J. Ackerman,2 B.P. Delisle1 1 University of Kentucky, Lexington, KY, 2Mayo Clinic, Rochester, MN. Background: Loss-of-function mutations in the KCNH2-ecoded Kv11.1 channel are 1 of the most common causes of long QT syndrome (LQTS). To date, dozens of different Kv11.1 missense mutations identified in patients with LQTS have been studied using heterologous expression. These studies show that 90% of these LQT-linked Kv11.1 mutations disrupt Kv11.1 trafficking and 5% alter Kv11.1 gating/ Kþ conduction. The purpose of this study was to determine whether this highly sensitive assay could be used to identify dysfunctional Kv11.1 mutations linked to sudden infant death syndrome (SIDS). Methods and Results: Postmortem genetic testing of 294 SIDS cases identified 9 nonsynonymous KCNH2 missense variants: E90K, R181Q, A190T, G294V, R791W, P967L, R1005W, R1047L, and Q1068R. R181Q, P967L, and R1047L were also identified in control subjects and previously shown to function similar to wild-type Kv11.1 (WT). Q1068R has also been identified in control subjects but alters Kv11.1 gating. We expressed E90K, G294V, R791W, and R1005W in HEK293 cells and studied their biochemical and biophysical properties using western blot and voltage clamping. Western blot analysis demonstrated that all of these variants undergo Golgi processing similar to WT, suggesting they do not disrupt Kv11.1 trafficking. Moreover, voltage clamping showed that they expressed similar amounts of macroscopic Kv11.1 current (IKv11.1) as cells expressing WT. Biophysical analysis demonstrated that R791W and R1005W altered Kv11.1 deactivation and activation, respectively. E90K and G294V channels had biophysical properties that were indistinguishable from WT.
2133 Conclusions: These data suggest that, unlike Kv11.1 missense mutations linked to LQTS, approximately two thirds of the Kv11.1 missense variants identified in SIDS cases are functional similar to WT and likely are benign. Although the remaining third traffic similar to WT, they alter Kv11.1 channel gating properties, which could increase an infant’s vulnerability to SIDS.
HEY2, A NOVEL SUSCEPTIBILITY GENE FOR BRUGADA SYNDROME, CONTROLS DEPOLARIZATION AND REPOLARIZATION GRADIENTS IN THE RVOT AND ACROSS THE VENTRICULAR WALL S. Podliesna,1 A.O. Verkerk,1 R. Wolswinkel,1 L. Beekman,1 J. Barc,1 M. Gessler,2 V.M. Christoffels,1 A.A. Wilde,1 C.A. Remme,1 C.R. Bezzina1 1 Academic Medical Center, Amsterdam, Netherlands, 2 Theodor-Boveri-Institute, Biocenter, University of Würzburg, Würzburg, Germany. Background: In a recent GWAS, we identified HEY2 as a novel susceptibility gene for Brugada syndrome. Studies in heterozygous Hey2 knockout mice (Hey2þ/–) demonstrated its role in mediating conduction velocity in the right ventricular outflow tract (RVOT). HEY2 is a transcriptional repressor putatively involved in establishing transmural gradients of genes across the ventricular wall. We investigated the effects of HEY2 on regional and transmural depolarization and repolarization patterns. Methods: Male adult Hey2þ/– mice and wild-type (WT) littermates were studied. Action potentials and membrane currents were measured in cardiomyocytes isolated from RVOT and subepicardial (EPI) and subendocardial (ENDO) regions of the right ventricle using the patchclamp technique. Real-time RT-PCR was performed on EPI and ENDO tissues from the left ventricle. Results: In WT cardiomyocytes, action potential duration (APD90) and upstroke velocity (Vmax) were significantly lower in WT-RVOT and WTEPI compared to WT-ENDO, demonstrating regional differences in depolarization and repolarization. Preliminary voltage-clamp experiments demonstrated higher transient outward Kþ current (Ito) densities in WTRVOT and WT-EPI compared to WT-ENDO, whereas steady-state Kþ currents were similar among the 3 sites. In line with this, Kcnd2 mRNA expression was significantly higher in WT-EPI compared to WT-ENDO. These regional and transmural differences were abrogated in Hey2þ/– hearts due to an increase in APD90 and Vmax specifically in EPI and RVOT. Ito density and Kcnd2 mRNA expression was significantly decreased in Hey2þ/– EPI but was unaffected in Hey2þ/– ENDO; hence a flattened transmural gradient of Ito and Kcnd2 was observed in Hey2þ/–. Ongoing experiments are focusing on the regional and transmural distribution of the Naþ current in WT vs Hey2þ/–. Conclusions: The transcriptional repressor HEY2, a novel susceptibility gene for Brugada syndrome, regulates APD90 and upstroke velocity in the RVOT and controls the transmural depolarization and repolarization gradient across the ventricular wall. These findings indicate that HEY2 modulates Brugada syndrome susceptibility through effects on both depolarization and repolarization processes.
GAIN-OF-FUNCTION MUTATION IN THE VOLTAGE-GATED Kþ CHANNEL BETA-2 SUBUNIT IS ASSOCIATED WITH BRUGADA SYNDROME V. Portero,1 S. Le Scouarnec,2 Z. Es-SalahLamoureux,1 S. Burel,1 J. Gourraud,1 S. Bonnaud,1 P. Lindenbaum,1 F. Simonet,1 J. Violleau,1 J. Sandoval-Tortosa,1 C. Scott,2 S. Chatel,1 G. Loussouarn,1 T. O’Hara,3 P. Mabo,4 C. Dina,1 H. Le Marec,3 J. Schott,1 V. Probst,1 I. Baró,1 C. Marionneau,1 F. Charpentier,1 R. Redon3 1 l’Institut du Thorax, Inserm, UMR 1087, CNRS, UMR 6291, Université de Nantes, Nantes, France, 2 The Wellcome Trust Sanger Institute, Hinxton,
Cambridge, United Kingdom, 3Johns Hopkins University, Baltimore, MD, 4University Hospital of Rennes, Rennes, France. Background: Brugada syndrome (BrS) is an inherited cardiac arrhythmia disorder characterized by ST-segment elevation on the electrocardiogram and associated with high risk of sudden cardiac death. Although mutations in the SCN5A gene have been causally related to BrS for around 20% of patients, the molecular mechanisms underlying this condition are still largely unknown. Methods and Results: We combined array-CGH, whole-exome sequencing, and linkage analysis to identify genetic variations likely causing BrS in a pedigree for which SCN5A mutations have been excluded. By this approach, we isolated 3 private missense variants co-segregating with cardiac rhythm anomalies within the pedigree. Among them, 1 variant resides in the KCNAB2 gene, which encodes Kvβ2, the voltage-gated Kþ channel beta-2 subunit (NM_003636: c.35G4A; NP_003627: p.Arg12Gln, R12Q). Kvβ2 is widely expressed in the human heart and has been shown to interact with the fast transient outward Kþ channel pore-forming subunit Kv4.3, increasing its current density. By screening the whole coding region of KCNAB2 in 190 unrelated patients with BrS, we found 2 additional rare missense variants (L13F and V114I). Patch-clamp experiments performed in COS-7 cells expressing both Kv4.3 and Kvβ2 revealed a significant increase in the density of Kv4.3-encoded current in the presence of 2 mutant forms of Kvβ2 (R12Q and L13F). Although biotinylation assays showed no differences in the cell surface expression of Kv4.3, the biotinylated fraction of Kvβ2-R12Q is significantly increased in comparison to wild-type Kvβ2. Conclusion: Our results indicate that gain-of-function mutations in KCNAB2 are associated with Brugada syndrome.
CONNEXIN43 (CX43) LACKING THE C-TERMINUS END REDUCES NAV1.5 CURRENT OF HL1 CELLS VIA A SEQUENCE RESIDING BETWEEN AMINO ACIDS 301 AND 361 L.W. Waring,1 E. Agullo-Pascual,2 M.S. Nielsen,1 M. Delmar1 1 University of Copenhagen, Copenhagen, Denmark, 2 New York University School of Medicine, New York, NY. Background: Recent studies suggest that connexins are a part of a protein interacting network regulating sodium channels independent of gap junction formation. We have shown that loss of the last 5 C-terminal amino acids of Cx43 (Cx43D378stop) reduces surface expression of the main cardiac sodium channel alpha subunit Nav1.5 at the ID in adult myocytes. In contrast, sodium current (INa) is normal when truncating Cx43 at M257, suggesting that an intermediate part of Cx43 has an inhibitory effect on sodium channel trafficking. The aim of the present study was to identify the inhibitory domain of the Cx43-CT. Methods and Results: Electrophysiology: HL1 cells carrying different Cx43 mutations were tested by whole-cell patch-clamp. INa was reduced in HL1 cells with a total knockdown of Cx43 and was restored by transient transfection of full-length Cx43, but not by Cx43 truncated at 378. The protective effect of the last 10 amino acids was independent of attachment to the Cx43 protein because the peptide SRPRPDDLEI partially restored INa. Truncations at 361 reduced INa, whereas larger truncations of the Cterminal (M257 and Y301stop) had no effect. These results indicate that the disruptive part of the CT resides between 301 and 361. Super-resolution fluorescence microscopy: Stable HL1 cell lines Cx43KD and HL1 WT were imaged by direct stochastic optical reconstruction microscopy (20-nm resolution). Cx43KD showed similar Nav1.5 mean cluster area compared to WT; however, the total number of Nav1.5 clusters was decreased (Po.0001). Moreover, Cx43KD showed decreased EB1 mean cluster area (Po.0001), suggesting separation of microtubule plus-end protein EB1 from N-cadherin–rich areas. Conclusions: Cx43KD resulted in reduced INa, which correlated with a reduced abundance of Nav1.5 clusters at N-cadherin–rich sites, likely due
LONG QT SYNDROME-ASSOCIATED CAVEOLIN-3 MUTATION F97C IMPARTS A LOSS-OF-FUNCTION EFFECT ON CARDIAC TRANSIENT OUTWARD POTASSIUM CURRENT (ITO) L. Tyan, W.M. Mesquitta, A.C. Grimes, R.C. Balijepalli, University of Wisconsin–Madison, Madison, WI. Background: Caveolae are discrete microdomains in cardiomyocytes that localize many ion channels and signaling proteins. Mutations in the CAV3 gene encoding Caveolin-3 (Cav3), a muscle-specific scaffolding protein integral to caveolae in the cardiomyocytes, have been associated with a congenital long QT syndrome (LQT9). The transient outward potassium current (Ito) is responsible for the rapid repolarization phase of the cardiac action potential (AP) and is one of the major determinants of the length of the QT interval. Kv4.2 and Kv4.3 channels generate the fast (Ito,f) and Kv1.4 channel mediates the slow (Ito,s) components of Ito. Previously we showed that LQTS-associated Cav3 mutations, F97C and S141R, prolong AP duration by increasing sodium channel late current, but whether these mutations impact the Ito is unknown. Methods: We determined the impact of LQTS-associated Cav3 mutations on the function of Kv4.2 and Kv4.3 channels using whole-cell patch-clamp analysis in HEK293 cells, by transiently co-expressing either wild-type (WT) Cav3, the LQT9-associated Cav3 mutations F97C or S141R, or a dominant-negative Cav3 mutation P104L. Co-immunoprecipitation and western blot analysis were performed to determine associations between Kv4.2 and/or Kv4.3 channels and Cav3. Results: Co-expression of F97C or P104 significantly reduced the peak Ikv4.2 and Ikv4.3 densities compared to cells transfected with either WT Cav-3 or vector controls. In contrast, co-expression of S141R did not significantly alter either Ikv4.2 or Ikv4.3. Analysis of activation and inactivation time constants of Kv4.2 and Kv4.3 channels revealed that F97C or P104L caused slower activation and slower inactivation of Ikv4.2 and slower activation of Ikv4.3. The inactivation time constant for Ikv4.3 was not affected. Furthermore, co-immunoprecipitation and western blot analysis revealed that Cav3 associates with Kv4.2 and Kv4.3 channels in mouse ventricular myocytes and transiently expressed HEK293 cells. Conclusions: Cav3 associates with Kv channels that form the Ito,f in cardiomyocytes. Furthermore, the LQT Cav-3 mutation F97C shows important loss-of-function effects on Ito, which will prolong repolarization and may contribute to arrhythmogenesis.
MICE THAT OVEREXPRESS A FRAMESHIFT MUTATION IN HUMAN NATRIURETIC PEPTIDE PRECURSOR A GENE EXHIBIT INCREASED ATRIAL FIBRILLATION BURDEN BUT NO ATRIAL STRUCTURAL REMODELING. E. Savio Galimberti, P. Kannankeril, K. Kor, S. Kupershmidt, S. Ansani, M. Blair, D. Darbar Vanderbilt University, Nashville, TN.
Heart Rhythm, Vol 11, No 11, November 2014 Background: A frameshift mutation in natriuretic peptide precursor A gene (NPPA) encoding a mutant atrial natriuretic peptide (ANP) has been linked with familial atrial fibrillation (AF). Mutant ANP shortened monophasic action potential duration and effective refractory period in retrogradely perfused rat hearts suggesting a potential mechanism for AF. We engineered 2 transgenic mouse lines that overexpress either mutant human NPPA (h-M) or human wild-type NPPA (h-WT) to test the hypothesis that h-M mice are more prone to develop AF. Methods: We determined circulating ANP levels and blood pressure (BP) in h-M, h-WT, and nontransgenic (NTG) B6D2 mice. To assess AF inducibility and burden, we conducted transesophageal pacing studies in anesthetized mice and isoproterenol challenge (ISO) in telemetry-implanted mice. To estimate atrial volumes and assess ventricular function we performed echocardiograms. Finally, we assessed atrial fibrosis in histologic slides dyed with Masson trichrome blue. Results: Circulating h-M-ANP level was 5-fold higher compared to h-WTANP and NTG-ANP (12.0 ⫾ 2.0 vs 4.0 ⫾ 1.0 vs 2.0 ⫾ 0.4 pg/mL, Po.01, n ¼ 5 mice/group), with cardiac tissue levels 6 orders of magnitude higher than blood levels. Mean BP was lower in h-M mice compared to hWT and NTG (68.8 ⫾ 1.0 vs 76.5 ⫾ 2.5 vs 85.1 ⫾ 1.6 mm Hg, Po.01, n ¼ 30 mice/group). h-M mice showed significantly increased AF burden/ animal than either h-WT or NTG (57.0 ⫾ 2.0 vs 5 ⫾ 1.0 vs 11.0 ⫾ 1.0 s, Po.05, n ¼ 5 mice/group). Also, 67% of h-M mice developed AF after ISO, which was more sustained than in NTG (21.3 ⫾ 3.0 vs 1.8 ⫾ 0.4 min, Po.01, n ¼ 3 mice/group), and h-WT mice did not develop AF. There were no changes in atrial volumes or fibrosis across groups. Cardiac output estimated by echo was preserved across groups (h-M: 28 ⫾ 2.0 vs h-WT: 32.2 ⫾ 1.4 vs NTG: 30.7 ⫾ 3.2 mL/min, n ¼ 5 mice/group). Although ejection fraction was similar in h-M (81.8 ⫾ 0.4%) and NTG (80.1 ⫾ 0.9%) and slightly decreased in h-WT (75.2 ⫾ 1.0 %, Po.01, n ¼ 5/group), all 3 groups were within normal range. Conclusions: Overexpression of human NPPA frameshift mutation in mice is associated with increased AF inducibility and burden. Absence of atrial fibrosis and preservation of ventricular function rule out global cardiomyopathy.
KCNH2 MUTATIONS THAT ALTER KV11.1 CHANNEL GATING ARE LINKED TO SUDDEN INFANT DEATH SYNDROME J.L. Smith,1 D.J. Tester,2 A.R. Reloj,1 D.E. Burgess,1 C. Hsu,1 M.J. Ackerman,2 B.P. Delisle1 1 University of Kentucky, Lexington, KY, 2Mayo Clinic, Rochester, MN. Background: Loss-of-function mutations in the KCNH2-ecoded Kv11.1 channel are 1 of the most common causes of long QT syndrome (LQTS). To date, dozens of different Kv11.1 missense mutations identified in patients with LQTS have been studied using heterologous expression. These studies show that 90% of these LQT-linked Kv11.1 mutations disrupt Kv11.1 trafficking and 5% alter Kv11.1 gating/ Kþ conduction. The purpose of this study was to determine whether this highly sensitive assay could be used to identify dysfunctional Kv11.1 mutations linked to sudden infant death syndrome (SIDS). Methods and Results: Postmortem genetic testing of 294 SIDS cases identified 9 nonsynonymous KCNH2 missense variants: E90K, R181Q, A190T, G294V, R791W, P967L, R1005W, R1047L, and Q1068R. R181Q, P967L, and R1047L were also identified in control subjects and previously shown to function similar to wild-type Kv11.1 (WT). Q1068R has also been identified in control subjects but alters Kv11.1 gating. We expressed E90K, G294V, R791W, and R1005W in HEK293 cells and studied their biochemical and biophysical properties using western blot and voltage clamping. Western blot analysis demonstrated that all of these variants undergo Golgi processing similar to WT, suggesting they do not disrupt Kv11.1 trafficking. Moreover, voltage clamping showed that they expressed similar amounts of macroscopic Kv11.1 current (IKv11.1) as cells expressing WT. Biophysical analysis demonstrated that R791W and R1005W altered Kv11.1 deactivation and activation, respectively. E90K and G294V channels had biophysical properties that were indistinguishable from WT.
2133 Conclusions: These data suggest that, unlike Kv11.1 missense mutations linked to LQTS, approximately two thirds of the Kv11.1 missense variants identified in SIDS cases are functional similar to WT and likely are benign. Although the remaining third traffic similar to WT, they alter Kv11.1 channel gating properties, which could increase an infant’s vulnerability to SIDS.
HEY2, A NOVEL SUSCEPTIBILITY GENE FOR BRUGADA SYNDROME, CONTROLS DEPOLARIZATION AND REPOLARIZATION GRADIENTS IN THE RVOT AND ACROSS THE VENTRICULAR WALL S. Podliesna,1 A.O. Verkerk,1 R. Wolswinkel,1 L. Beekman,1 J. Barc,1 M. Gessler,2 V.M. Christoffels,1 A.A. Wilde,1 C.A. Remme,1 C.R. Bezzina1 1 Academic Medical Center, Amsterdam, Netherlands, 2 Theodor-Boveri-Institute, Biocenter, University of Würzburg, Würzburg, Germany. Background: In a recent GWAS, we identified HEY2 as a novel susceptibility gene for Brugada syndrome. Studies in heterozygous Hey2 knockout mice (Hey2þ/–) demonstrated its role in mediating conduction velocity in the right ventricular outflow tract (RVOT). HEY2 is a transcriptional repressor putatively involved in establishing transmural gradients of genes across the ventricular wall. We investigated the effects of HEY2 on regional and transmural depolarization and repolarization patterns. Methods: Male adult Hey2þ/– mice and wild-type (WT) littermates were studied. Action potentials and membrane currents were measured in cardiomyocytes isolated from RVOT and subepicardial (EPI) and subendocardial (ENDO) regions of the right ventricle using the patchclamp technique. Real-time RT-PCR was performed on EPI and ENDO tissues from the left ventricle. Results: In WT cardiomyocytes, action potential duration (APD90) and upstroke velocity (Vmax) were significantly lower in WT-RVOT and WTEPI compared to WT-ENDO, demonstrating regional differences in depolarization and repolarization. Preliminary voltage-clamp experiments demonstrated higher transient outward Kþ current (Ito) densities in WTRVOT and WT-EPI compared to WT-ENDO, whereas steady-state Kþ currents were similar among the 3 sites. In line with this, Kcnd2 mRNA expression was significantly higher in WT-EPI compared to WT-ENDO. These regional and transmural differences were abrogated in Hey2þ/– hearts due to an increase in APD90 and Vmax specifically in EPI and RVOT. Ito density and Kcnd2 mRNA expression was significantly decreased in Hey2þ/– EPI but was unaffected in Hey2þ/– ENDO; hence a flattened transmural gradient of Ito and Kcnd2 was observed in Hey2þ/–. Ongoing experiments are focusing on the regional and transmural distribution of the Naþ current in WT vs Hey2þ/–. Conclusions: The transcriptional repressor HEY2, a novel susceptibility gene for Brugada syndrome, regulates APD90 and upstroke velocity in the RVOT and controls the transmural depolarization and repolarization gradient across the ventricular wall. These findings indicate that HEY2 modulates Brugada syndrome susceptibility through effects on both depolarization and repolarization processes.
GAIN-OF-FUNCTION MUTATION IN THE VOLTAGE-GATED Kþ CHANNEL BETA-2 SUBUNIT IS ASSOCIATED WITH BRUGADA SYNDROME V. Portero,1 S. Le Scouarnec,2 Z. Es-SalahLamoureux,1 S. Burel,1 J. Gourraud,1 S. Bonnaud,1 P. Lindenbaum,1 F. Simonet,1 J. Violleau,1 J. Sandoval-Tortosa,1 C. Scott,2 S. Chatel,1 G. Loussouarn,1 T. O’Hara,3 P. Mabo,4 C. Dina,1 H. Le Marec,3 J. Schott,1 V. Probst,1 I. Baró,1 C. Marionneau,1 F. Charpentier,1 R. Redon3 1 l’Institut du Thorax, Inserm, UMR 1087, CNRS, UMR 6291, Université de Nantes, Nantes, France, 2 The Wellcome Trust Sanger Institute, Hinxton,
Cambridge, United Kingdom, 3Johns Hopkins University, Baltimore, MD, 4University Hospital of Rennes, Rennes, France. Background: Brugada syndrome (BrS) is an inherited cardiac arrhythmia disorder characterized by ST-segment elevation on the electrocardiogram and associated with high risk of sudden cardiac death. Although mutations in the SCN5A gene have been causally related to BrS for around 20% of patients, the molecular mechanisms underlying this condition are still largely unknown. Methods and Results: We combined array-CGH, whole-exome sequencing, and linkage analysis to identify genetic variations likely causing BrS in a pedigree for which SCN5A mutations have been excluded. By this approach, we isolated 3 private missense variants co-segregating with cardiac rhythm anomalies within the pedigree. Among them, 1 variant resides in the KCNAB2 gene, which encodes Kvβ2, the voltage-gated Kþ channel beta-2 subunit (NM_003636: c.35G4A; NP_003627: p.Arg12Gln, R12Q). Kvβ2 is widely expressed in the human heart and has been shown to interact with the fast transient outward Kþ channel pore-forming subunit Kv4.3, increasing its current density. By screening the whole coding region of KCNAB2 in 190 unrelated patients with BrS, we found 2 additional rare missense variants (L13F and V114I). Patch-clamp experiments performed in COS-7 cells expressing both Kv4.3 and Kvβ2 revealed a significant increase in the density of Kv4.3-encoded current in the presence of 2 mutant forms of Kvβ2 (R12Q and L13F). Although biotinylation assays showed no differences in the cell surface expression of Kv4.3, the biotinylated fraction of Kvβ2-R12Q is significantly increased in comparison to wild-type Kvβ2. Conclusion: Our results indicate that gain-of-function mutations in KCNAB2 are associated with Brugada syndrome.
CONNEXIN43 (CX43) LACKING THE C-TERMINUS END REDUCES NAV1.5 CURRENT OF HL1 CELLS VIA A SEQUENCE RESIDING BETWEEN AMINO ACIDS 301 AND 361 L.W. Waring,1 E. Agullo-Pascual,2 M.S. Nielsen,1 M. Delmar1 1 University of Copenhagen, Copenhagen, Denmark, 2 New York University School of Medicine, New York, NY. Background: Recent studies suggest that connexins are a part of a protein interacting network regulating sodium channels independent of gap junction formation. We have shown that loss of the last 5 C-terminal amino acids of Cx43 (Cx43D378stop) reduces surface expression of the main cardiac sodium channel alpha subunit Nav1.5 at the ID in adult myocytes. In contrast, sodium current (INa) is normal when truncating Cx43 at M257, suggesting that an intermediate part of Cx43 has an inhibitory effect on sodium channel trafficking. The aim of the present study was to identify the inhibitory domain of the Cx43-CT. Methods and Results: Electrophysiology: HL1 cells carrying different Cx43 mutations were tested by whole-cell patch-clamp. INa was reduced in HL1 cells with a total knockdown of Cx43 and was restored by transient transfection of full-length Cx43, but not by Cx43 truncated at 378. The protective effect of the last 10 amino acids was independent of attachment to the Cx43 protein because the peptide SRPRPDDLEI partially restored INa. Truncations at 361 reduced INa, whereas larger truncations of the Cterminal (M257 and Y301stop) had no effect. These results indicate that the disruptive part of the CT resides between 301 and 361. Super-resolution fluorescence microscopy: Stable HL1 cell lines Cx43KD and HL1 WT were imaged by direct stochastic optical reconstruction microscopy (20-nm resolution). Cx43KD showed similar Nav1.5 mean cluster area compared to WT; however, the total number of Nav1.5 clusters was decreased (Po.0001). Moreover, Cx43KD showed decreased EB1 mean cluster area (Po.0001), suggesting separation of microtubule plus-end protein EB1 from N-cadherin–rich areas. Conclusions: Cx43KD resulted in reduced INa, which correlated with a reduced abundance of Nav1.5 clusters at N-cadherin–rich sites, likely due