Jason D. Foell

Mutant Caveolin-3 Induces Persistent Late Sodium Current and Is Associated With Long-QT Syndrome

Background— Congenital long-QT syndrome (LQTS) is a primary arrhythmogenic syndrome stemming from perturbed cardiac repolarization. LQTS, which affects ≈1 in 3000 persons, is 1 of the most common causes of autopsy-negative sudden death in the young. Since the sentinel discovery of cardiac channel gene mutations in LQTS in 1995, hundreds of mutations in 8 LQTS susceptibility genes have been identified. All 8 LQTS genotypes represent primary cardiac channel defects (ie, ion channelopathy) except LQT4, which is a functional channelopathy because of mutations in ankyrin-B. Approximately 25% of LQTS remains unexplained pathogenetically. We have pursued a “final common pathway” hypothesis to elicit novel LQTS-susceptibility genes. With the recent observation that the LQT3-associated, SCN5A-encoded cardiac sodium channel localizes in caveolae, which are known membrane microdomains whose major component in the striated muscle is caveolin-3, we hypothesized that mutations in caveolin-3 may represent a novel pathogenetic mechanism for LQTS. Methods and Results— Using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing, we performed open reading frame/splice site mutational analysis on CAV3 in 905 unrelated patients referred for LQTS genetic testing. CAV3 mutations were engineered by site-directed mutagenesis and the molecular phenotype determined by transient heterologous expression into cell lines that stably express the cardiac sodium channel hNav1.5. We identified 4 novel mutations in CAV3-encoded caveolin-3 that were absent in >1000 control alleles. Electrophysiological analysis of sodium current in HEK293 cells stably expressing hNav1.5 and transiently transfected with wild-type and mutant caveolin-3 demonstrated that mutant caveolin-3 results in a 2- to 3-fold increase in late sodium current compared with wild-type caveolin-3. Our observations are similar to the increased late sodium current associated with LQT3-associated SCN5A mutations. Conclusions— The present study reports the first CAV3 mutations in subjects with LQTS, and we provide functional data demonstrating a gain-of-function increase in late sodium current.

Reduction in density of transverse tubules and L-type Ca2+ channels in canine tachycardia-induced heart failure

OBJECTIVE Persistent supraventricular tachycardia leads to the development of a dilated cardiomyopathy with impairment of excitation-contraction (EC) coupling. Since the initial trigger for EC coupling in ventricular muscle is the influx of Ca(2+) through L-type Ca(2+) channels (I(Ca)) in the transverse tubules (T-tubules), we determined if the density of the T-tubule system and L-type Ca(2+) channels change in canine tachycardia pacing-induced cardiomyopathy. METHODS Confocal imaging of isolated ventricular myocytes stained with the membrane dye Di-8-ANEPPS was used to image the T-tubule system, and standard whole-cell patch clamp techniques were used to measure I(Ca) and intramembrane charge movement. RESULTS A complex staining pattern of interconnected tubules including prominent transverse components spaced every approximately 1.6 microm was present in control ventricular myocytes, but failing cells demonstrated a far less regular T-tubule system with a relative loss of T-tubules. In confocal optical slices, the average % of the total cell area staining for T-tubules decreased from 11.5+/-0.4 in control to 8.7+/-0.4% in failing cells (P<0.001). Whole-cell patch clamp studies revealed that I(Ca) density was unchanged. Since whole-cell I(Ca) is due to both the number of channels as well as the functional properties of those channels, we measured intramembrane charge movement as an assay for changes in channel number. The saturating amount of charge that moves due to gating of L-type Ca(2+) channels, Q(on,max), was decreased from 6.5+/-0.6 in control to 2.8+/-0.3 fC/pF in failing myocytes (P<0.001). CONCLUSIONS Cellular remodeling in heart failure results in decreased density of T-tubules and L-type Ca(2+) channels, which contribute to abnormal EC coupling.

Small GTPase Determinants for the Golgi Processing and Plasmalemmal Expression of Human Ether-a-go-go Related (hERG) K+ Channels

The pro-arrhythmic Long QT syndrome (LQT) is linked to 10 different genes (LQT1–10). Approximately 40% of genotype-positive LQT patients have LQT2, which is characterized by mutations in the human ether-a-go-go related gene (hERG). hERG encodes the voltage-gated K+ channel α-subunits that form the pore of the rapidly activating delayed rectifier K+ current in the heart. The purpose of this study was to elucidate the mechanisms that regulate the intracellular transport or trafficking of hERG, because trafficking is impaired for about 90% of LQT2 missense mutations. Protein trafficking is regulated by small GTPases. To identify the small GTPases that are critical for hERG trafficking, we coexpressed hERG and dominant negative (DN) GTPase mutations in HEK293 cells. The GTPases Sar1 and ARF1 regulate the endoplasmic reticulum (ER) export of proteins in COPII and COPI vesicles, respectively. Expression of DN Sar1 inhibited the Golgi processing of hERG, decreased hERG current (IhERG) by 85% (n ≥ 8 cells per group, *, p < 0.01), and reduced the plasmalemmal staining of hERG. The coexpression of DN ARF1 had relatively small effects on hERG trafficking. Surprisingly, the coexpression of DN Rab11B, which regulates the endosomal recycling, inhibited the Golgi processing of hERG, decreased IhERG by 79% (n ≥ 8 cells per group; *, p < 0.01), and reduced the plasmalemmal staining of hERG. These data suggest that hERG undergoes ER export in COPII vesicles and endosomal recycling prior to being processed in the Golgi. We conclude that hERG trafficking involves a pathway between the ER and endosomal compartments that influences expression in the plasmalemma.

Kv11.1 (ERG1) K + Channels Localize in Cholesterol and Sphingolipid Enriched Membranes and Are Modulated by Membrane Cholesterol

The localization of ion channels to specific membrane microdomains can impact the functional properties of channels and their role in cellular physiology. We determined the membrane localization of human Kv11.1 (hERG1) α -subunit protein, which underlies the rapidly activating, delayed rectifier K+ current (IKr) in the heart. Immunocytochemistry and membrane fractionation using discontinuous sucrose density gradients of adult canine ventricular tissue showed that Kv11.1 channel protein localized to both the cell surface and T-tubular sarcolemma. Furthermore, density gradient membrane fractionation using detergent (Triton X-100) and non-detergent (OptiPrep) methods from canine ventricular myocytes or HEK293 cells demonstrated that Kv11.1 protein, along with MiRP1 and Kv7.1 (KCNQ1) proteins, localize in cholesterol and sphingolipid enriched membrane fractions. In HEK293 cells, Kv11.1 channels, but not long QT-associated mutant G601S-Kv11.1 channels, also localized to cholesterol and sphingolipid enriched membrane fractions. Depletion of membrane cholesterol from HEK293 cells expressing Kv11.1 channels using methyl-Mβ -cyclodextrin (Mβ CD) caused a positive shift of the voltage dependence of activation and an acceleration of deactivation kinetics of Kv11.1 current (IKv11.1). Cholesterol loading of HEK293 cells reduced the steep voltage dependence of IKv11.1 activation and accelerated the inactivation kinetics of IKv11.1. Incubation of neonatal mouse myocytes in Mβ CD also accelerated the deactivation kinetics of IKr. We conclude that Kv11.1 protein localizes in cholesterol and sphingolipid enriched membranes and that membrane cholesterol can modulate IKv11.1 and IKr.

Small GTPase Rab11b regulates degradation of surface membrane L-type Cav1.2 channels

L-type Ca(2+) channels (LTCCs) play a critical role in Ca(2+)-dependent signaling processes in a variety of cell types. The number of functional LTCCs at the plasma membrane strongly influences the strength and duration of Ca(2+) signals. Recent studies demonstrated that endosomal trafficking provides a mechanism for dynamic changes in LTCC surface membrane density. The purpose of the current study was to determine whether the small GTPase Rab11b, a known regulator of endosomal recycling, impacts plasmalemmal expression of Ca(v)1.2 LTCCs. Disruption of endogenous Rab11b function with a dominant negative Rab11b S25N mutant led to a significant 64% increase in peak L-type Ba(2+) current (I(Ba,L)) in human embryonic kidney (HEK)293 cells. Short-hairpin RNA (shRNA)-mediated knockdown of Rab11b also significantly increased peak I(Ba,L) by 66% compared when with cells transfected with control shRNA, whereas knockdown of Rab11a did not impact I(Ba,L). Rab11b S25N led to a 1.7-fold increase in plasma membrane density of hemagglutinin epitope-tagged Ca(v)1.2 expressed in HEK293 cells. Cell surface biotinylation experiments demonstrated that Rab11b S25N does not significantly impact anterograde trafficking of LTCCs to the surface membrane but rather slows degradation of plasmalemmal Ca(v)1.2 channels. We further demonstrated Rab11b expression in ventricular myocardium and showed that Rab11b S25N significantly increases peak I(Ba,L) by 98% in neonatal mouse cardiac myocytes. These findings reveal a novel role for Rab11b in limiting, rather than promoting, the plasma membrane expression of Ca(v)1.2 LTCCs in contrast to its effects on other ion channels including human ether-a-go-go-related gene (hERG) K(+) channels and cystic fibrosis transmembrane conductance regulator. This suggests Rab11b differentially regulates the trafficking of distinct cargo and extends our understanding of how endosomal transport impacts the functional expression of LTCCs.

Robust L-type calcium current expression following heterozygous knockout of the Cav1.2 gene in adult mouse heart

Non‐technical summary  Appropriate regulation of ion channel expression is critical for the maintenance of both electrical stability and normal contractile function in the heart. A classic way to study the robustness of biological systems is to examine the effects of changes in gene dosage. We have studied how the heart responds to changes in the L‐type calcium channel gene dosage. Homozygous Cav1.2 knockout in the adult heart is lethal, without compensatory responses in expression of other calcium channel genes. Following heterozygous knockout, Cav1.2 mRNA levels are not buffered, Cav1.2 membrane protein levels are partly buffered and L‐type calcium current expression is relatively well buffered. These data are consistent with a passive model of Cav1.2 biosynthesis that includes saturated steps, which act to buffer Cav1.2 protein and L‐type calcium current expression. The results suggest that there is little or no homeostatic regulation of calcium current expression in either heterozygous or homozygous knockout mice.

Blocking the L-type Ca2+ Channel With a Gem: A Paradigm for a More Specific Ca2+ Channel Blocker

Ca2+ channel blockers have been an important part of the cardiovascular pharmacological armamentarium for more than 2 decades. These agents were developed as antianginals and antihypertensives, but their indications have expanded to include treatment of certain arrhythmias because of their atrioventricular (AV) nodal blocking properties. Interestingly, the development of these compounds largely preceded our knowledge of the molecular composition and detailed functional properties of voltage-gated Ca2+ channels. In fact, Ca2+ channel blockers were critical in defining the distinct class of voltage-dependent Ca2+ channels referred to as L-type Ca2+ channels, which can be found in cardiac myocytes, skeletal myocytes, vascular smooth muscle cells, neurons, and endocrine cells among other cells. Despite this broad distribution of L-type Ca2+ channels, Ca2+ channel blockers have proven useful agents because they exhibit pharmacological specificity for vascular smooth muscle and cardiac muscle. This specificity is attributable to a variety of factors including the voltage and use-dependent blocking properties of these agents, subtle differences in the sensitivity of distinct isoforms of L-type Ca2+ channels present in different tissues, and the tissue distribution of the drugs. In addition, different classes of Ca2+ channel blockers vary in their relative potency to block vascular smooth muscle Ca2+ channels (antihypertensive/antianginal properties) relative to their block of AV nodal Ca2+ channels (antiarrhythmic properties). Alas, the specificity is not perfect, and so these agents can bring with them undesired side effects including constipation, peripheral edema, dizziness, and headache. In addition, problems can arise from the overlap of vascular smooth muscle and cardiac blocking properties. For example, if one wants to control a rapid heart rate in a hypotensive patient with atrial fibrillation by blocking AV nodal Ca2+ channels, accompanying vasodilation from vascular smooth muscle blockade can be problematic. Alternatively, treating angina …Ca2+ channel blockers have been an important part of the cardiovascular pharmacological armamentarium for more than 2 decades. These agents were developed as antianginals and antihypertensives, but their indications have expanded to include treatment of certain arrhythmias because of their atrioventricular (AV) nodal blocking properties. Interestingly, the development of these compounds largely preceded our knowledge of the molecular composition and detailed functional properties of voltage-gated Ca2+ channels. In fact, Ca2+ channel blockers were critical in defining the distinct class of voltage-dependent Ca2+ channels referred to as L-type Ca2+ channels, which can be found in cardiac myocytes, skeletal myocytes, vascular smooth muscle cells, neurons, and endocrine cells among other cells. Despite this broad distribution of L-type Ca2+ channels, Ca2+ channel blockers have proven useful agents because they exhibit pharmacological specificity for vascular smooth muscle and cardiac muscle. This specificity is attributable to a variety of factors including the voltage and use-dependent blocking properties of these agents, subtle differences in the sensitivity of distinct isoforms of L-type Ca2+ channels present in different tissues, and the tissue distribution of the drugs. In addition, different classes of Ca2+ channel blockers vary in their relative potency to block vascular smooth muscle Ca2+ channels (antihypertensive/antianginal properties) relative to their block of AV nodal Ca2+ channels (antiarrhythmic properties). Alas, the specificity is not perfect, and so these agents can bring with them undesired side effects including constipation, peripheral edema, dizziness, and headache. In addition, problems can arise from the overlap of vascular smooth muscle and cardiac blocking properties. For example, if one wants to control a rapid heart rate in a hypotensive patient with atrial fibrillation by blocking AV nodal Ca2+ channels, accompanying vasodilation from vascular smooth muscle blockade can be problematic. Alternatively, treating angina …

Predictability of lesion durability for AF ablation using phased radiofrequency: Power, temperature, and duration impact creation of transmural lesions

BACKGROUND Long-term clinical outcomes for atrial fibrillation ablation depend on the creation of durable transmural lesions during pulmonary vein isolation and on substrate modification. Focal conventional radiofrequency (RF) ablation studies have demonstrated that tissue temperature and power are important factors for lesion formation. However, the impact and predictability of temperature and power on contiguous, transmural lesion formation with a phased RF system has not been described. OBJECTIVE The purpose of this study was to determine the sensitivity, specificity, and predictability of power and temperature to create contiguous, transmural lesions with the temperature-controlled, multielectrode phased RF PVAC GOLD catheter. METHODS Single ablations with the PVAC GOLD catheter were performed in the superior vena cava of 22 pigs. Ablations from 198 PVAC GOLD electrodes were evaluated by gross examination and histopathology for lesion transmurality and contiguity. Lesions were compared to temperature and power data from the phased RF GENius generator. Effective contact was defined as electrodes with a temperature of ≥50°C and a power of ≥3 W. RESULTS Eighty-five percent (168 of 198) of the lesions were transmural and 79% (106 of 134) were contiguous. Electrode analysis showed that >30 seconds of effective contact identified transmural lesions with 85% sensitivity (95% confidence interval [CI] 78%-89%), 93% specificity (95% CI 76%-99%), and 99% positive predictive value (95% CI 94%-100%). Sensitivity for lesion contiguity was 95% (95% CI 89%-98%), with 62% specificity (95% CI 42%-78%) and 90% positive predictive value (95% CI 83%-95%). No char or coagulum was observed on the catheter or tissue. CONCLUSION PVAC GOLD safely, effectively, and predictably creates transmural and contiguous lesions.

Blocking the L-type Ca2+ Channel With a Gem

Ca2+ channel blockers have been an important part of the cardiovascular pharmacological armamentarium for more than 2 decades. These agents were developed as antianginals and antihypertensives, but their indications have expanded to include treatment of certain arrhythmias because of their atrioventricular (AV) nodal blocking properties. Interestingly, the development of these compounds largely preceded our knowledge of the molecular composition and detailed functional properties of voltage-gated Ca2+ channels. In fact, Ca2+ channel blockers were critical in defining the distinct class of voltage-dependent Ca2+ channels referred to as L-type Ca2+ channels, which can be found in cardiac myocytes, skeletal myocytes, vascular smooth muscle cells, neurons, and endocrine cells among other cells. Despite this broad distribution of L-type Ca2+ channels, Ca2+ channel blockers have proven useful agents because they exhibit pharmacological specificity for vascular smooth muscle and cardiac muscle. This specificity is attributable to a variety of factors including the voltage and use-dependent blocking properties of these agents, subtle differences in the sensitivity of distinct isoforms of L-type Ca2+ channels present in different tissues, and the tissue distribution of the drugs. In addition, different classes of Ca2+ channel blockers vary in their relative potency to block vascular smooth muscle Ca2+ channels (antihypertensive/antianginal properties) relative to their block of AV nodal Ca2+ channels (antiarrhythmic properties). Alas, the specificity is not perfect, and so these agents can bring with them undesired side effects including constipation, peripheral edema, dizziness, and headache. In addition, problems can arise from the overlap of vascular smooth muscle and cardiac blocking properties. For example, if one wants to control a rapid heart rate in a hypotensive patient with atrial fibrillation by blocking AV nodal Ca2+ channels, accompanying vasodilation from vascular smooth muscle blockade can be problematic. Alternatively, treating angina …Ca2+ channel blockers have been an important part of the cardiovascular pharmacological armamentarium for more than 2 decades. These agents were developed as antianginals and antihypertensives, but their indications have expanded to include treatment of certain arrhythmias because of their atrioventricular (AV) nodal blocking properties. Interestingly, the development of these compounds largely preceded our knowledge of the molecular composition and detailed functional properties of voltage-gated Ca2+ channels. In fact, Ca2+ channel blockers were critical in defining the distinct class of voltage-dependent Ca2+ channels referred to as L-type Ca2+ channels, which can be found in cardiac myocytes, skeletal myocytes, vascular smooth muscle cells, neurons, and endocrine cells among other cells. Despite this broad distribution of L-type Ca2+ channels, Ca2+ channel blockers have proven useful agents because they exhibit pharmacological specificity for vascular smooth muscle and cardiac muscle. This specificity is attributable to a variety of factors including the voltage and use-dependent blocking properties of these agents, subtle differences in the sensitivity of distinct isoforms of L-type Ca2+ channels present in different tissues, and the tissue distribution of the drugs. In addition, different classes of Ca2+ channel blockers vary in their relative potency to block vascular smooth muscle Ca2+ channels (antihypertensive/antianginal properties) relative to their block of AV nodal Ca2+ channels (antiarrhythmic properties). Alas, the specificity is not perfect, and so these agents can bring with them undesired side effects including constipation, peripheral edema, dizziness, and headache. In addition, problems can arise from the overlap of vascular smooth muscle and cardiac blocking properties. For example, if one wants to control a rapid heart rate in a hypotensive patient with atrial fibrillation by blocking AV nodal Ca2+ channels, accompanying vasodilation from vascular smooth muscle blockade can be problematic. Alternatively, treating angina …

Blocking the L-type Ca 2+ Channel With a Gem

Ca2+ channel blockers have been an important part of the cardiovascular pharmacological armamentarium for more than 2 decades. These agents were developed as antianginals and antihypertensives, but their indications have expanded to include treatment of certain arrhythmias because of their atrioventricular (AV) nodal blocking properties. Interestingly, the development of these compounds largely preceded our knowledge of the molecular composition and detailed functional properties of voltage-gated Ca2+ channels. In fact, Ca2+ channel blockers were critical in defining the distinct class of voltage-dependent Ca2+ channels referred to as L-type Ca2+ channels, which can be found in cardiac myocytes, skeletal myocytes, vascular smooth muscle cells, neurons, and endocrine cells among other cells. Despite this broad distribution of L-type Ca2+ channels, Ca2+ channel blockers have proven useful agents because they exhibit pharmacological specificity for vascular smooth muscle and cardiac muscle. This specificity is attributable to a variety of factors including the voltage and use-dependent blocking properties of these agents, subtle differences in the sensitivity of distinct isoforms of L-type Ca2+ channels present in different tissues, and the tissue distribution of the drugs. In addition, different classes of Ca2+ channel blockers vary in their relative potency to block vascular smooth muscle Ca2+ channels (antihypertensive/antianginal properties) relative to their block of AV nodal Ca2+ channels (antiarrhythmic properties). Alas, the specificity is not perfect, and so these agents can bring with them undesired side effects including constipation, peripheral edema, dizziness, and headache. In addition, problems can arise from the overlap of vascular smooth muscle and cardiac blocking properties. For example, if one wants to control a rapid heart rate in a hypotensive patient with atrial fibrillation by blocking AV nodal Ca2+ channels, accompanying vasodilation from vascular smooth muscle blockade can be problematic. Alternatively, treating angina …Ca2+ channel blockers have been an important part of the cardiovascular pharmacological armamentarium for more than 2 decades. These agents were developed as antianginals and antihypertensives, but their indications have expanded to include treatment of certain arrhythmias because of their atrioventricular (AV) nodal blocking properties. Interestingly, the development of these compounds largely preceded our knowledge of the molecular composition and detailed functional properties of voltage-gated Ca2+ channels. In fact, Ca2+ channel blockers were critical in defining the distinct class of voltage-dependent Ca2+ channels referred to as L-type Ca2+ channels, which can be found in cardiac myocytes, skeletal myocytes, vascular smooth muscle cells, neurons, and endocrine cells among other cells. Despite this broad distribution of L-type Ca2+ channels, Ca2+ channel blockers have proven useful agents because they exhibit pharmacological specificity for vascular smooth muscle and cardiac muscle. This specificity is attributable to a variety of factors including the voltage and use-dependent blocking properties of these agents, subtle differences in the sensitivity of distinct isoforms of L-type Ca2+ channels present in different tissues, and the tissue distribution of the drugs. In addition, different classes of Ca2+ channel blockers vary in their relative potency to block vascular smooth muscle Ca2+ channels (antihypertensive/antianginal properties) relative to their block of AV nodal Ca2+ channels (antiarrhythmic properties). Alas, the specificity is not perfect, and so these agents can bring with them undesired side effects including constipation, peripheral edema, dizziness, and headache. In addition, problems can arise from the overlap of vascular smooth muscle and cardiac blocking properties. For example, if one wants to control a rapid heart rate in a hypotensive patient with atrial fibrillation by blocking AV nodal Ca2+ channels, accompanying vasodilation from vascular smooth muscle blockade can be problematic. Alternatively, treating angina …