John W. Kyle

Two human paramyotonia congenita mutations have opposite effects on lidocaine block of Na+ channels expressed in a mammalian cell line.

1. Two mutant human skeletal muscle voltage‐gated Na+ channel alpha‐subunits (hSkM1), with mutations found in patients with hereditary paramyotonia congenita (T1313M on the III‐IV linker and R1448C on the outside of S4 of repeat IV), and wild‐type hSkM1 channels were expressed in a human embryonic kidney cell lines (tsA201) using recombinant cDNA. 2. Compared with wild‐type, both mutants exhibited altered inactivation phenotypes. Current decay was slowed for both, but voltage‐dependent availability from inactivation was shifted in the negative direction for R1448C and in the positive direction for T1313M. 3. The hypothesis that a local anaesthetic, lidocaine (lignocaine), binds primarily to the inactivated state to block the channel was reassessed by testing lidocaine block of these two mutants and the wild‐type channel. 4. T1313M showed reduced phasic block, but R1448C showed increased phasic block for trains of depolarizations. 5. Rest block (from ‐120 mV) was increased for R1448C (IC50 approximately equal to 0.2 mM) and decreased for T1313M (IC50 approximately equal to 1.3 mM) compared with wild‐type (IC50 approximately 0.5 mM), but these differences were diminished at a holding potential of ‐150 mV, suggesting that the differences were caused by binding to the inactivated state rather than a different affinity of lidocaine for the resting state. 6. Inactivated state affinity measured from lidocaine‐induced shifts in voltage‐dependent availability was reduced for T1313M (Kd = 63 microM) but little changed for R1448C (Kd = 14 microM) compared with wild‐type (Kd = 11 microM). Two pulse recovery protocols showed faster recovery from lidocaine block for T1313M and slower recovery for R1448C. Together these accounted for the opposite effects on lidocaine phasic block observed for the mutant channels. 7. Neither mutation is located at a putative lidocaine binding site in domain 4 S6, yet both affected lidocaine block. The data suggest that R1448C altered phasic lidocaine block mainly through altered kinetics, but T1313M altered block through a change in affinity for the inactivated state. These findings have implications for drug therapy of paramyotonia congenita, and also provide an insight into structural requirements for drug affinity.

Mechanisms of Sudden Cardiac Death Oxidants and Metabolism

Ventricular arrhythmia is the leading cause of sudden cardiac death (SCD). Deranged cardiac metabolism and abnormal redox state during cardiac diseases foment arrhythmogenic substrates through direct or indirect modulation of cardiac ion channel/transporter function. This review presents current evidence on the mechanisms linking metabolic derangement and excessive oxidative stress to ion channel/transporter dysfunction that predisposes to ventricular arrhythmias and SCD. Because conventional antiarrhythmic agents aiming at ion channels have proven challenging to use, targeting arrhythmogenic metabolic changes and redox imbalance may provide novel therapeutics to treat or prevent life-threatening arrhythmias and SCD.

Intrinsic lidocaine affinity for Na channels expressed in Xenopus oocytes depends on α (hH1 vs. rSkM1) and β1 subunits

Objective: The affinity of lidocaine for the α-subunit of the Na channel has been reported to be greater for heart than for non-heart α-subunits, and also to be no different. Lidocaine block has a complex voltage dependence caused by a higher affinity for the inactivated state over the resting state. Inactivation kinetics, however, depend upon the α-subunit isoform and the presence of the auxiliary β1-subunit and will affect measures of block. Methods: We studied the voltage dependence of lidocaine block of Na currents by a two microelectrode voltage clamp in oocytes injected with RNA for the Na channel α-subunits of human heart (hH1a) or a rat skeletal muscle (rSkM1) alone, or coexpressed with the β1-subunit. Results: The midpoints of availability for a 25-s conditioning potential in control solutions were −65 mV for rSkM1, −50 for rSkM1+β1, −78 mV for hH1a and −76 for hH1a+β1. The Kd of tonic lidocaine block was measured at −90, −100, −110, −120 and −130 mV in the same oocytes. The apparent Kd for both isoforms ±β1 became greater with more negative holding potentials, but tended to reach different plateaus at −130 mV (Kd=2128 μM for rSkM1, 1760 μM for rSkM1+β1, 433 for hH1a, and 887 μM for hH1a+β1). Inactivated state affinities, assessed by fitting the shift in the Boltzmann midpoint of the availability relationship to the modulated receptor model, were 4 μM for rSkM1, 1 μM for rSkM1+β1, 7 μM for hH1a and 9 μM for hH1a+β1. Conclusion: The heart Na channel α-subunits expressed in oocytes have an intrinsically higher rest state affinity for lidocaine compared to rSkM1 after the voltage- and state dependence of block are considered. Coexpression with β1 modestly increased the rest affinity of lidocaine for rSkM1, but had the opposite effect for hH1a.

An East Asian Common Variant Vinculin P.Asp841His Was Associated With Sudden Unexplained Nocturnal Death Syndrome in the Chinese Han Population

Background We have identified the cardiomyopathy‐susceptibility gene vinculin (VCL) mutation M94I may account for a sudden unexplained nocturnal death syndrome (SUNDS) case. We addressed whether VCL common variant D841H is associated with SUNDS. Methods and Results In 8 of 120 SUNDS cases, we detected an East Asian common VCL variant p.Asp841His (D841H). Comparing the H841 allele frequency of the general population in the local database (15 of 1818) with SUNDS victims (10 of 240) gives an odds ratio for SUNDS of 5.226 (95% CI, 2.321, 11.769). The VCL‐D841H variant was engineered and either coexpressed with cardiac sodium channel (SCN5A) in HEK293 cells or overexpressed in human induced pluripotent stem‐cell–derived cardiomyocytes to examine its effects on sodium channel function using the whole‐cell patch‐clamp method. In HEK293 cells, under physiological pH conditions (pH 7.4), D841H caused a 29% decrease in peak IN a amplitude compared to wild type (WT), whereas under acidotic conditions (pH 7.0), D841H decreased further to 43% along with significant negative shift in inactivation compared to WT at pH 7.4. In induced pluripotent stem‐cell‐derived cardiomyocytes, similar effects of D841H on IN a were observed. VCL colocalized with SCN5A at the intercalated disk in human cardiomyocytes. VCL was also confirmed to directly interact with SCN5A, and VCL‐D841H did not disrupt the association of VCL and SCN5A. Conclusions A VCL common variant was genetically and biophysically associated with Chinese SUNDS. The aggravation of loss of function of SCN5A caused by VCL‐D841H under acidosis supports that nocturnal sleep respiratory disorders with acidosis may play a key role in the pathogenesis of SUNDS.

Late I(Na) in the Heart: Physiology, Pathology, and Pathways.

In this issue of Circulation , Glynn and colleagues1 make an important contribution to our understanding of the physiological and pathophysiological roles of late sodium current ( I Na) in the heart, with a focus on a key pathways regulating late I Na amplitude. They conducted well-designed and detailed studies with 2 new genetically engineered mouse lines: an S571A mouse that ablates phosphorylation by Ca2+/calmodulin-dependent kinase II (CaMKII)2 selectively at serine 571 (S571) in the cardiac Na channel pore-forming protein Scn5a and an S571E mouse that mimics phosphorylation at S571. S571 was shown previously to be a target for phosphorylation by CaMKII, and this phosphorylation enhanced late I Na.2 The present studies in “knock-in” mice expressing either S571A or S571E have distinct advantages over earlier studies in heterologous expression systems, including cultured myocyte models, because they allow the study of whole-animal and organ phenotypes and cellular and molecular biophysical properties in a more native environment. These new in vivo studies reveal that, despite the extensive network of CaMKII targets, phosphorylation of S571 selectively regulates late I Na and, in particular, enhanced late I Na in failing heart. Article see p 567 Peak I Na is the large inward current flowing mainly through the cardiac Na+ channel pore formed by Scn5a, which is part of a larger sodium channel macromolecular complex. Members of this macromolecular complex act to localize the complex and to regulate I Na.3 With the onset of the action potential (AP) in the myocardium, the peak I Na rapidly rises and decays to nearly zero over several milliseconds. This I Na spike underlies excitability and conduction in working myocardium and the Purkinje conduction system. In contrast to peak I Na, late I Na is a small inward current, usually …In this issue of Circulation , Glynn and colleagues1 make an important contribution to our understanding of the physiological and pathophysiological roles of late sodium current ( I Na) in the heart, with a focus on a key pathways regulating late I Na amplitude. They conducted well-designed and detailed studies with 2 new genetically engineered mouse lines: an S571A mouse that ablates phosphorylation by Ca2+/calmodulin-dependent kinase II (CaMKII)2 selectively at serine 571 (S571) in the cardiac Na channel pore-forming protein Scn5a and an S571E mouse that mimics phosphorylation at S571. S571 was shown previously to be a target for phosphorylation by CaMKII, and this phosphorylation enhanced late I Na.2 The present studies in “knock-in” mice expressing either S571A or S571E have distinct advantages over earlier studies in heterologous expression systems, including cultured myocyte models, because they allow the study of whole-animal and organ phenotypes and cellular and molecular biophysical properties in a more native environment. These new in vivo studies reveal that, despite the extensive network of CaMKII targets, phosphorylation of S571 selectively regulates late I Na and, in particular, enhanced late I Na in failing heart. Article see p 567 Peak I Na is the large inward current flowing mainly through the cardiac Na+ channel pore formed by Scn5a, which is part of a larger sodium channel macromolecular complex. Members of this macromolecular complex act to localize the complex and to regulate I Na.3 With the onset of the action potential (AP) in the myocardium, the peak I Na rapidly rises and decays to nearly zero over several milliseconds. This I Na spike underlies excitability and conduction in working myocardium and the Purkinje conduction system. In contrast to peak I Na, late I Na is a small inward current, usually …

Mouse ERG K+ Channel Clones Reveal Differences in Protein Trafficking and Function

Background The mouse ether‐a‐go‐go‐related gene 1a (mERG1a, mKCNH2) encodes mERG K+ channels in mouse cardiomyocytes. The mERG channels and their human analogue, hERG channels, conduct IKr. Mutations in hERG channels reduce IKr to cause congenital long‐QT syndrome type 2, mostly by decreasing surface membrane expression of trafficking‐deficient channels. Three cDNA sequences were originally reported for mERG channels that differ by 1 to 4 amino acid residues (mERG‐London, mERG‐Waterston, and mERG‐Nie). We characterized these mERG channels to test the postulation that they would differ in their protein trafficking and biophysical function, based on previous findings in long‐QT syndrome type 2. Methods and Results The 3 mERG and hERG channels were expressed in HEK293 cells and neonatal mouse cardiomyocytes and were studied using Western blot and whole‐cell patch clamp. We then compared our findings with the recent sequencing results in the Welcome Trust Sanger Institute Mouse Genomes Project (WTSIMGP). Conclusions First, the mERG‐London channel with amino acid substitutions in regions of highly ordered structure is trafficking deficient and undergoes temperature‐dependent and pharmacological correction of its trafficking deficiency. Second, the voltage dependence of channel gating would be different for the 3 mERG channels. Third, compared with the WTSIMGP data set, the mERG‐Nie clone is likely to represent the wild‐type mouse sequence and physiology. Fourth, the WTSIMGP analysis suggests that substrain‐specific sequence differences in mERG are a common finding in mice. These findings with mERG channels support previous findings with hERG channel structure–function analyses in long‐QT syndrome type 2, in which sequence changes in regions of highly ordered structure are likely to result in abnormal protein trafficking.

Transgenic overexpression of the SUR2A-55 splice variant in mouse heart reduces infract size and promotes protective mitochondrial function

ATP-sensitive potassium channels found in both the sarcolemma (sarcKATP) and mitochondria (mitoKATP) of cardiomyocytes are important mediators of cardioprotection during ischemic heart disease. Sulfonylurea receptor isoforms (SUR2), encoded by Abcc9, an ATP-binding cassette family member, form regulatory subunits of the sarcKATP channel and are also thought to regulate mitoKATP channel activity. A short-form splice variant of SUR2 (SUR2A-55) was previously shown to target mitochondria and display diaxoxide and ATP insensitive KATP activity when co-expressed with the inward rectifier channels Kir6.2 and Kir6.1. We hypothesized that mice with cardiac specific overexpression of SUR2A-55 would mediate cardioprotection from ischemia by altering mitoKATP properties. Mice overexpressing SUR2A-55 (TGSUR2A-55) in cardiomyocytes were generated and showed no significant difference in echocardiographic measured chamber dimension, percent fractional shortening, heart to body weight ratio, or gross histologic features compared to normal mice at 11–14 weeks of age. TGSUR2A-55 had improved hemodynamic functional recovery and smaller infarct size after ischemia reperfusion injury compared to WT mice in an isolated hanging heart model. The mitochondrial membrane potential of TGSUR2A-55 mice was less sensitive to ATP, diazoxide, and Ca2+ loading. These data suggest that the SUR2A-55 splice variant favorably affects mitochondrial function leading to cardioprotection. These data support a role for the regulation of mitoKATP activity by SUR2A-55.

Late INa in the Heart

In this issue of Circulation , Glynn and colleagues1 make an important contribution to our understanding of the physiological and pathophysiological roles of late sodium current ( I Na) in the heart, with a focus on a key pathways regulating late I Na amplitude. They conducted well-designed and detailed studies with 2 new genetically engineered mouse lines: an S571A mouse that ablates phosphorylation by Ca2+/calmodulin-dependent kinase II (CaMKII)2 selectively at serine 571 (S571) in the cardiac Na channel pore-forming protein Scn5a and an S571E mouse that mimics phosphorylation at S571. S571 was shown previously to be a target for phosphorylation by CaMKII, and this phosphorylation enhanced late I Na.2 The present studies in “knock-in” mice expressing either S571A or S571E have distinct advantages over earlier studies in heterologous expression systems, including cultured myocyte models, because they allow the study of whole-animal and organ phenotypes and cellular and molecular biophysical properties in a more native environment. These new in vivo studies reveal that, despite the extensive network of CaMKII targets, phosphorylation of S571 selectively regulates late I Na and, in particular, enhanced late I Na in failing heart. Article see p 567 Peak I Na is the large inward current flowing mainly through the cardiac Na+ channel pore formed by Scn5a, which is part of a larger sodium channel macromolecular complex. Members of this macromolecular complex act to localize the complex and to regulate I Na.3 With the onset of the action potential (AP) in the myocardium, the peak I Na rapidly rises and decays to nearly zero over several milliseconds. This I Na spike underlies excitability and conduction in working myocardium and the Purkinje conduction system. In contrast to peak I Na, late I Na is a small inward current, usually …In this issue of Circulation , Glynn and colleagues1 make an important contribution to our understanding of the physiological and pathophysiological roles of late sodium current ( I Na) in the heart, with a focus on a key pathways regulating late I Na amplitude. They conducted well-designed and detailed studies with 2 new genetically engineered mouse lines: an S571A mouse that ablates phosphorylation by Ca2+/calmodulin-dependent kinase II (CaMKII)2 selectively at serine 571 (S571) in the cardiac Na channel pore-forming protein Scn5a and an S571E mouse that mimics phosphorylation at S571. S571 was shown previously to be a target for phosphorylation by CaMKII, and this phosphorylation enhanced late I Na.2 The present studies in “knock-in” mice expressing either S571A or S571E have distinct advantages over earlier studies in heterologous expression systems, including cultured myocyte models, because they allow the study of whole-animal and organ phenotypes and cellular and molecular biophysical properties in a more native environment. These new in vivo studies reveal that, despite the extensive network of CaMKII targets, phosphorylation of S571 selectively regulates late I Na and, in particular, enhanced late I Na in failing heart. Article see p 567 Peak I Na is the large inward current flowing mainly through the cardiac Na+ channel pore formed by Scn5a, which is part of a larger sodium channel macromolecular complex. Members of this macromolecular complex act to localize the complex and to regulate I Na.3 With the onset of the action potential (AP) in the myocardium, the peak I Na rapidly rises and decays to nearly zero over several milliseconds. This I Na spike underlies excitability and conduction in working myocardium and the Purkinje conduction system. In contrast to peak I Na, late I Na is a small inward current, usually …

Late I Na in the Heart

In this issue of Circulation , Glynn and colleagues1 make an important contribution to our understanding of the physiological and pathophysiological roles of late sodium current ( I Na) in the heart, with a focus on a key pathways regulating late I Na amplitude. They conducted well-designed and detailed studies with 2 new genetically engineered mouse lines: an S571A mouse that ablates phosphorylation by Ca2+/calmodulin-dependent kinase II (CaMKII)2 selectively at serine 571 (S571) in the cardiac Na channel pore-forming protein Scn5a and an S571E mouse that mimics phosphorylation at S571. S571 was shown previously to be a target for phosphorylation by CaMKII, and this phosphorylation enhanced late I Na.2 The present studies in “knock-in” mice expressing either S571A or S571E have distinct advantages over earlier studies in heterologous expression systems, including cultured myocyte models, because they allow the study of whole-animal and organ phenotypes and cellular and molecular biophysical properties in a more native environment. These new in vivo studies reveal that, despite the extensive network of CaMKII targets, phosphorylation of S571 selectively regulates late I Na and, in particular, enhanced late I Na in failing heart. Article see p 567 Peak I Na is the large inward current flowing mainly through the cardiac Na+ channel pore formed by Scn5a, which is part of a larger sodium channel macromolecular complex. Members of this macromolecular complex act to localize the complex and to regulate I Na.3 With the onset of the action potential (AP) in the myocardium, the peak I Na rapidly rises and decays to nearly zero over several milliseconds. This I Na spike underlies excitability and conduction in working myocardium and the Purkinje conduction system. In contrast to peak I Na, late I Na is a small inward current, usually …In this issue of Circulation , Glynn and colleagues1 make an important contribution to our understanding of the physiological and pathophysiological roles of late sodium current ( I Na) in the heart, with a focus on a key pathways regulating late I Na amplitude. They conducted well-designed and detailed studies with 2 new genetically engineered mouse lines: an S571A mouse that ablates phosphorylation by Ca2+/calmodulin-dependent kinase II (CaMKII)2 selectively at serine 571 (S571) in the cardiac Na channel pore-forming protein Scn5a and an S571E mouse that mimics phosphorylation at S571. S571 was shown previously to be a target for phosphorylation by CaMKII, and this phosphorylation enhanced late I Na.2 The present studies in “knock-in” mice expressing either S571A or S571E have distinct advantages over earlier studies in heterologous expression systems, including cultured myocyte models, because they allow the study of whole-animal and organ phenotypes and cellular and molecular biophysical properties in a more native environment. These new in vivo studies reveal that, despite the extensive network of CaMKII targets, phosphorylation of S571 selectively regulates late I Na and, in particular, enhanced late I Na in failing heart. Article see p 567 Peak I Na is the large inward current flowing mainly through the cardiac Na+ channel pore formed by Scn5a, which is part of a larger sodium channel macromolecular complex. Members of this macromolecular complex act to localize the complex and to regulate I Na.3 With the onset of the action potential (AP) in the myocardium, the peak I Na rapidly rises and decays to nearly zero over several milliseconds. This I Na spike underlies excitability and conduction in working myocardium and the Purkinje conduction system. In contrast to peak I Na, late I Na is a small inward current, usually …