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1.
Physiol Rep ; 11(15): e15779, 2023 08.
Article in English | MEDLINE | ID: mdl-37537144

ABSTRACT

Remodeling of cardiac t-tubules in normal and pathophysiological conditions is an important process contributing to the functional performance of the heart. While it is well documented that deterioration of t-tubule network associated with various pathological conditions can be reversed under certain conditions, the mechanistic understanding of the recovery process is essentially lacking. Accordingly, in this study we investigated some aspects of the recovery of t-tubules after experimentally-induced detubulation. T-tubules of isolated mouse ventricular myocytes were first sealed using osmotic shock approach, and their recovery under various experimental conditions was then characterized using electrophysiologic and imaging techniques. The data show that t-tubule recovery is a strongly temperature-dependent process involving reopening of previously collapsed t-tubular segments. T-tubule recovery is slowed by (1) metabolic inhibition of cells, (2) reducing influx of extracellular Ca2+ as well as by (3) both stabilization and disruption of microtubules. Overall, the data show that t-tubule recovery is a highly dynamic process involving several central intracellular structures and processes and lay the basis for more detailed investigations in this area.


Subject(s)
Myocytes, Cardiac , Sarcolemma , Mice , Animals , Myocytes, Cardiac/metabolism , Sarcolemma/metabolism , Calcium/metabolism , Calcium Signaling/physiology
2.
JCI Insight ; 6(3)2021 02 08.
Article in English | MEDLINE | ID: mdl-33411695

ABSTRACT

Loss-of-function (LOF) variants in SCN1B, encoding voltage-gated sodium channel ß1 subunits, are linked to human diseases with high risk of sudden death, including developmental and epileptic encephalopathy and cardiac arrhythmia. ß1 Subunits modulate the cell-surface localization, gating, and kinetics of sodium channel pore-forming α subunits. They also participate in cell-cell and cell-matrix adhesion, resulting in intracellular signal transduction, promotion of cell migration, calcium handling, and regulation of cell morphology. Here, we investigated regulated intramembrane proteolysis (RIP) of ß1 by BACE1 and γ-secretase and show that ß1 subunits are substrates for sequential RIP by BACE1 and γ-secretase, resulting in the generation of a soluble intracellular domain (ICD) that is translocated to the nucleus. Using RNA sequencing, we identified a subset of genes that are downregulated by ß1-ICD overexpression in heterologous cells but upregulated in Scn1b-null cardiac tissue, which lacks ß1-ICD signaling, suggesting that the ß1-ICD may normally function as a molecular brake on gene transcription in vivo. We propose that human disease variants resulting in SCN1B LOF cause transcriptional dysregulation that contributes to altered excitability. Moreover, these results provide important insights into the mechanism of SCN1B-linked channelopathies, adding RIP-excitation coupling to the multifunctionality of sodium channel ß1 subunits.


Subject(s)
Voltage-Gated Sodium Channel beta-1 Subunit/metabolism , Amyloid Precursor Protein Secretases/metabolism , Animals , Aspartic Acid Endopeptidases/metabolism , Cell Membrane/metabolism , Cells, Cultured , Cricetulus , Excitation Contraction Coupling/genetics , Excitation Contraction Coupling/physiology , Gene Expression , HEK293 Cells , Humans , Loss of Function Mutation , Mice , Mice, Knockout , Myocytes, Cardiac/metabolism , Proteolysis , RNA Splicing Factors/genetics , RNA Splicing Factors/metabolism , Signal Transduction , Voltage-Gated Sodium Channel beta-1 Subunit/deficiency , Voltage-Gated Sodium Channel beta-1 Subunit/genetics
3.
Am J Physiol Heart Circ Physiol ; 319(2): H410-H421, 2020 08 01.
Article in English | MEDLINE | ID: mdl-32648820

ABSTRACT

Cardiac t tubules undergo significant remodeling in various pathological and experimental conditions, which can be associated with mechanical or osmotic stress. In particular, it has been shown that removal of hyposmotic stress can lead to sealing of t tubules. However, the mechanisms underlying the sealing process remain essentially unknown. In this study we used dextran trapping assay to demonstrate that in adult mouse cardiomyocytes, t-tubular sealing can also be induced by hyperosmotic challenge and that both hypo- and hyperosmotic sealing display a clear threshold behavior requiring ≈100 mosmol/L minimal stress. Importantly, during both hypo- and hyperosmotic challenges, the sealing of t tubules occurs only during the shrinking phase. Analysis of the time course of t-tubular remodeling following removal of hyposmotic stress shows that t tubules become sealed essentially instantly, well before any significant reduction in cell size can be observed. Overall, the data support the hypothesis that the critical event in the process of t-tubular sealing during osmotic challenges is detachment (peeling) of the membrane from the underlying cytoskeleton due to suprathreshold stress.NEW & NOTEWORTHY This study provides new insights into how t-tubular membranes respond to osmotic forces. In particular, the data show that osmotically induced sealing of cardiac t tubules is a threshold phenomenon initiated by detachment of t-tubular membrane from the underlying cytoskeleton. The findings are consistent with the hypothesis that final sealing of t tubules is driven by negative hydrostatic intracellular pressure coincident with cell shrinking.


Subject(s)
Cell Membrane/pathology , Cell Size , Cytoskeleton/pathology , Myocytes, Cardiac/pathology , Osmotic Pressure , Vacuoles/pathology , Animals , Cell Membrane/metabolism , Cytoskeleton/metabolism , Female , Male , Mice, Inbred C57BL , Myocytes, Cardiac/metabolism , Time Factors , Vacuoles/metabolism
4.
Front Physiol ; 9: 1516, 2018.
Article in English | MEDLINE | ID: mdl-30483142

ABSTRACT

Efficient excitation-contraction coupling in ventricular myocytes depends critically on the presence of the t-tubular network. It has been recently demonstrated that cholesterol, a major component of the lipid bilayer, plays an important role in long-term maintenance of the integrity of t-tubular system although mechanistic understanding of underlying processes is essentially lacking. Accordingly, in this study we investigated the contribution of membrane cholesterol to t-tubule remodeling in response to acute hyposmotic stress. Experiments were performed using isolated left ventricular cardiomyocytes from adult mice. Depletion and restoration of membrane cholesterol was achieved by applying methyl-ß-cyclodextrin (MßCD) and water soluble cholesterol (WSC), respectively, and t-tubule remodeling in response to acute hyposmotic stress was assessed using fluorescent dextran trapping assay and by measuring t-tubule dependent IK1 tail current (IK1,tail). The amount of dextran trapped in t-tubules sealed in response to stress was significantly increased when compared to control cells, and reintroduction of cholesterol to cells treated with MßCD restored the amount of trapped dextran to control values. Alternatively, application of WSC to normal cells significantly reduced the amount of trapped dextran further suggesting the protective effect of cholesterol. Importantly, modulation of membrane cholesterol (without osmotic stress) led to significant changes in various parameters of IK1, tail strongly suggesting significant but essentially hidden remodeling of t-tubules prior to osmotic stress. Results of this study demonstrate that modulation of the level of membrane cholesterol has significant effects on the susceptibility of cardiac t-tubules to acute hyposmotic stress.

5.
Biophys J ; 114(2): 437-449, 2018 01 23.
Article in English | MEDLINE | ID: mdl-29401441

ABSTRACT

Cardiac t-tubules (TTs) form a network of complex surface membrane invaginations that is essential for proper excitation-contraction coupling. Although electron and optical microscopy studies provided a wealth of important information about the structure of TTs, assessing their functional properties remains a challenge. In this study, we investigated the diffusional accessibility of TTs in intact isolated adult mouse ventricular myocytes using, to our knowledge, a novel fluorescence-based assay. In this approach, a small part of TTs is first locally filled with fluorescent dextran and then its diffusion out of TTs is monitored after rapid removal of extracellular dextran. In normal cells, diffusion of 3 kDa dextran is characterized by an average time constant of 3.9 ± 1.2 s with the data ranging from 1.8 to 10.5 s. The data are consistent with essentially free diffusion of dextran in TTs although measurable contribution of binding is also evident. TT fluorescence is abolished in cells treated with high concentration of formamide or after hyposmotic stress. Importantly, the assay we use allows for quantitative, repetitive measurements of subtle dynamic changes in TT structure of the same cell that are not possible to observe with other approaches. In particular, dextran diffusion rate decreases two-to-threefold during cell swelling, suggesting significant structural remodeling of TTs. Computer modeling shows that diffusional accessibility and electrical properties of TTs are primarily determined by the constrictions and dilations of individual TTs and that, from a functional perspective, TTs cannot be considered as a network of cylinders of the same average diameter. Constriction/dilation model of cardiac TTs is in a quantitative agreement with previous high-resolution microscopy studies of TT structure and alternative measurements of diffusional and electrical time constants of TTs. The data also show that the apparent electrical length constant of cardiac TTs is likely several-fold smaller than that estimated in earlier studies.


Subject(s)
Cell Membrane/metabolism , Electrophysiological Phenomena , Mechanical Phenomena , Animals , Biomechanical Phenomena , Dextrans/metabolism , Diffusion , Female , Male , Mice , Models, Cardiovascular , Myocytes, Cardiac/cytology , Osmosis
6.
Am J Physiol Heart Circ Physiol ; 311(1): H229-38, 2016 07 01.
Article in English | MEDLINE | ID: mdl-27208165

ABSTRACT

Cardiac t-tubules are critical for efficient excitation-contraction coupling but become significantly remodeled during various stress conditions. However, the mechanisms by which t-tubule remodeling occur are poorly understood. Recently, we demonstrated that recovery of mouse ventricular myocytes after hyposmotic shock is associated with t-tubule sealing. In this study, we found that the application of Small Membrane Permeable Molecules (SMPM) such as DMSO, formamide and acetamide upon washout of hyposmotic solution significantly reduced the amount of extracellular dextran trapped within sealed t-tubules. The SMPM protection displayed sharp biphasic concentration dependence that peaks at ∼140 mM leading to >3- to 4-fold reduction in dextran trapping. Consistent with these data, detailed analysis of the effects of DMSO showed that the magnitude of normalized inward rectifier tail current (IK1,tail), an electrophysiological marker of t-tubular integrity, was increased ∼2-fold when hyposmotic stress was removed in the presence of 1% DMSO (∼140 mM). Analysis of dynamics of cardiomyocytes shrinking during resolution of hyposmotic stress revealed only minor increase in shrinking rate in the presence of 1% DMSO, and cell dimensions returned fully to prestress values in both control and DMSO groups. Application and withdrawal of 10% DMSO in the absence of preceding hyposmotic shock induced classical t-tubule sealing. This suggests that the biphasic concentration dependence originated from an increase in secondary t-tubule sealing when high SMPM concentrations are removed. Overall, the data suggest that SMPM protect against sealing of t-tubules following hyposmotic stress, likely through membrane modification and essentially independent of their osmotic effects.


Subject(s)
Acetamides/pharmacology , Cell Membrane Permeability , Cell Membrane/drug effects , Dimethyl Sulfoxide/pharmacology , Formamides/pharmacology , Myocytes, Cardiac/drug effects , Osmotic Pressure , Acetamides/chemistry , Acetamides/metabolism , Animals , Cell Membrane/metabolism , Dextrans/metabolism , Dimethyl Sulfoxide/chemistry , Dimethyl Sulfoxide/metabolism , Dose-Response Relationship, Drug , Excitation Contraction Coupling/drug effects , Female , Formamides/chemistry , Formamides/metabolism , Intermediate-Conductance Calcium-Activated Potassium Channels/drug effects , Intermediate-Conductance Calcium-Activated Potassium Channels/metabolism , Male , Membrane Potentials , Mice, Inbred C57BL , Molecular Weight , Myocardial Contraction/drug effects , Myocytes, Cardiac/metabolism
7.
Am J Physiol Heart Circ Physiol ; 301(5): H1984-95, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21890686

ABSTRACT

Cardiac ventricular myocytes possess an extensive t-tubular system that facilitates the propagation of membrane potential across the cell body. It is well established that ionic currents at the restricted t-tubular space may lead to significant changes in ion concentrations, which, in turn, may affect t-tubular membrane potential. In this study, we used the whole cell patch-clamp technique to study accumulation and depletion of t-tubular potassium by measuring inward rectifier potassium tail currents (I(K1,tail)), and inward rectifier potassium current (I(K1)) "inactivation". At room temperatures and in the absence of Mg(2+) ions in pipette solution, the amplitude of I(K1,tail) measured ~10 min after the establishment of whole cell configuration was reduced by ~18%, but declined nearly twofold in the presence of 1 mM cyanide. At ~35°C I(K1,tail) was essentially preserved in intact cells, but its amplitude declined by ~85% within 5 min of cell dialysis, even in the absence of cyanide. Intracellular Mg(2+) ions played protective role at all temperatures. Decline of I(K1,tail) was accompanied by characteristic changes in its kinetics, as well as by changes in the kinetics of I(K1) inactivation, a marker of depletion of t-tubular K(+). The data point to remodeling of t tubules as the primary reason for the observed effects. Consistent with this, detubulation of myocytes using formamide-induced osmotic stress significantly reduced I(K1,tail), as well as the inactivation of inward I(K1). Overall, the data provide strong evidence that changes in t tubule volume/structure may occur on a short time scale in response to various types of stress.


Subject(s)
Energy Metabolism , Heart Ventricles/metabolism , Myocytes, Cardiac/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Potassium/metabolism , Stress, Physiological , Ventricular Remodeling , Animals , Cyanides/pharmacology , Energy Metabolism/drug effects , Female , Formamides/pharmacology , Heart Ventricles/drug effects , Heart Ventricles/pathology , Ion Transport , Kinetics , Magnesium/metabolism , Male , Membrane Potentials , Mice , Mice, Inbred C57BL , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/pathology , Osmotic Pressure , Patch-Clamp Techniques , Stress, Physiological/drug effects , Temperature , Ventricular Remodeling/drug effects
8.
Pflugers Arch ; 460(5): 839-49, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20676672

ABSTRACT

Kir2 subunits form channels that underlie classical strongly inwardly rectifying potassium currents. While homomeric Kir2 channels display a number of distinct and physiologically important properties, the functional properties of heteromeric Kir2 assemblies, as well as the stoichiometries and the arrangements of Kir2 subunits in native channels, remain largely unknown. Therefore, we have implemented a concatemeric approach, whereby all four cloned Kir2 subunits were linked in tandem, in order to study the effects of Kir2.1 and Kir2.2 heteromerization on properties of the resulting channels. Kir2.2 subunits contributed stronger to single-channel conductance than Kir2.1 subunits, and channels containing two or more Kir2.2 subunits displayed conductances indistinguishable from that of a Kir2.2 homomeric channel. In contrast, single-channel kinetics was a more discriminating property. The open times were significantly shorter in Kir2.2 channels compared with Kir2.1 channels and decreased nearly proportionally to the number of Kir2.2 subunits in the heteromeric channel. Similarly, the sensitivity to block by barium also depended on the proportions of Kir2.1 to Kir2.2 subunits. Overall, the results showed that Kir2.1 and Kir2.2 subunits exert neither a dominant nor an anomalous effect on any of the properties of heteromeric channels. The data highlight opportunities and challenges of using differential properties of Kir2 channels in deciphering the subunit composition of native inwardly rectifying potassium currents.


Subject(s)
Potassium Channels, Inwardly Rectifying/physiology , Animals , Barium/pharmacology , Cloning, Molecular , HEK293 Cells , Humans , Mice , Potassium Channels, Inwardly Rectifying/drug effects
9.
J Mol Cell Cardiol ; 48(1): 45-54, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19703462

ABSTRACT

Cardiac I(K1) and I(KACh) are the major potassium currents displaying classical strong inward rectification, a unique property that is critical for their roles in cardiac excitability. In the last 15 years, research on I(K1) and I(KACh) has been propelled by the cloning of the underlying inwardly rectifying potassium (Kir) channels, the discovery of the molecular mechanism of strong rectification and the linking of a number of disorders of cardiac excitability to defects in genes encoding Kir channels. Disease-causing mutations in Kir genes have been shown experimentally to affect one or more of the following channel properties: structure, assembly, trafficking, and regulation, with the ultimate effect of a gain- or a loss-of-function of the channel. It is now established that I(K1) and I(KACh) channels are heterotetramers of Kir2 and Kir3 subunits, respectively. Each homomeric Kir channel has distinct biophysical and regulatory properties, and individual Kir subunits often display different patterns of regional, cellular, and membrane distribution. These differences are thought to underlie important variations in the physiological properties of I(K1) and I(KACh). It has become increasingly clear that the contribution of I(K1) and I(KACh) channels to cardiac electrical activity goes beyond their long recognized role in the stabilization of resting membrane potential and shaping the late phase of action potential repolarization in individual myocytes but extends to being critical elements determining the overall electrical stability of the heart.


Subject(s)
Heart/physiology , Myocardium/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Action Potentials/genetics , Action Potentials/physiology , Animals , Heart/physiopathology , Humans , Membrane Potentials/genetics , Membrane Potentials/physiology , Models, Biological , Myocardium/pathology , Potassium Channels, Inwardly Rectifying/genetics
10.
J Phys Chem B ; 113(32): 11179-85, 2009 Aug 13.
Article in English | MEDLINE | ID: mdl-19606833

ABSTRACT

It has long been recognized that cationic nanoparticles induce cell membrane permeability. Recently, it has been found that cationic nanoparticles induce the formation and/or growth of nanoscale holes in supported lipid bilayers. In this paper, we show that noncytotoxic concentrations of cationic nanoparticles induce 30-2000 pA currents in 293A (human embryonic kidney) and KB (human epidermoid carcinoma) cells, consistent with a nanoscale defect such as a single hole or group of holes in the cell membrane ranging from 1 to 350 nm(2) in total area. Other forms of nanoscale defects, including the nanoparticle porating agents adsorbing onto or intercalating into the lipid bilayer, are also consistent; although the size of the defect must increase to account for any reduction in ion conduction, as compared to a water channel. An individual defect forming event takes 1-100 ms, while membrane resealing may occur over tens of seconds. Patch-clamp data provide direct evidence for the formation of nanoscale defects in living cell membranes. The cationic polymer data are compared and contrasted with patch-clamp data obtained for an amphiphilic phenylene ethynylene antimicrobial oligomer (AMO-3), a small molecule that is proposed to make well-defined 3.4 nm holes in lipid bilayers. Here, we observe data that are consistent with AMO-3 making approximately 3 nm holes in living cell membranes.


Subject(s)
Cations , Cell Membrane/metabolism , Nanoparticles , Cell Line , Humans , Nanotechnology
11.
Physiol Genomics ; 33(3): 312-22, 2008 May 13.
Article in English | MEDLINE | ID: mdl-18334547

ABSTRACT

Relaxation abnormalities are prevalent in heart failure and contribute to clinical outcomes. Disruption of Ca2+ homeostasis in heart failure delays relaxation by prolonging the intracellular Ca2+ transient. We sought to speed cardiac relaxation in vivo by cardiac-directed transgene expression of parvalbumin (Parv), a cytosolic Ca2+ buffer normally expressed in fast-twitch skeletal muscle. A key feature of Parv's function resides in its Ca2+/Mg2+ binding affinities that account for delayed Ca2+ buffering in response to the intracellular Ca2+ transient. Cardiac Parv expression decreased sarcoplasmic reticulum Ca2+ content without otherwise altering intracellular Ca2+ homeostasis. At high physiological mouse heart rates in vivo, Parv modestly accelerated relaxation without affecting cardiac morphology or systolic function. Ex vivo pacing of the isolated heart revealed a marked heart rate dependence of Parv's delayed Ca2+ buffering effects on myocardial performance. As the pacing frequency was lowered (7 to 2.5 Hz), the relaxation rates increased in Parv hearts. However, as pacing rates approached the dynamic range in humans, Parv hearts demonstrated decreased contractility, consistent with Parv buffering systolic Ca2+. Mathematical modeling and in vitro studies provide the underlying mechanism responsible for the frequency-dependent fractional Ca2+ buffering action of Parv. Future studies directed toward refining the dose and frequency-response relationships of Parv in the heart or engineering novel Parv-based Ca2+ buffers with modified Mg2+ and Ca2+ affinities to limit systolic Ca2+ buffering may hold promise for the development of new therapies to remediate relaxation abnormalities in heart failure.


Subject(s)
Calcium/metabolism , Gene Expression/physiology , Heart Rate/physiology , Myocardium/metabolism , Parvalbumins/biosynthesis , Parvalbumins/genetics , Animals , Buffers , Calcium Signaling/genetics , Calcium Signaling/physiology , Cardiac Pacing, Artificial , Gene Expression/genetics , Heart Rate/genetics , Homeostasis , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Cardiovascular , Myocardial Contraction/genetics , Myocardial Contraction/physiology , Myocytes, Cardiac/metabolism , Organ Culture Techniques , Organ Specificity/genetics , Rats , Sarcoplasmic Reticulum/metabolism , Transgenes
12.
J Mol Cell Cardiol ; 43(5): 636-47, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17884088

ABSTRACT

In neurons, voltage-gated sodium channel beta subunits regulate the expression levels, subcellular localization, and electrophysiological properties of sodium channel alpha subunits. However, the contribution of beta subunits to sodium channel function in heart is poorly understood. We examined the role of beta1 in cardiac excitability using Scn1b null mice. Compared to wildtype mice, electrocardiograms recorded from Scn1b null mice displayed longer RR intervals and extended QT(c) intervals, both before and after autonomic block. In acutely dissociated ventricular myocytes, loss of beta1 expression resulted in a approximately 1.6-fold increase in both peak and persistent sodium current while channel gating and kinetics were unaffected. Na(v)1.5 expression increased in null myocytes approximately 1.3-fold. Action potential recordings in acutely dissociated ventricular myocytes showed slowed repolarization, supporting the extended QT(c) interval. Immunostaining of individual myocytes or ventricular sections revealed no discernable alterations in the localization of sodium channel alpha or beta subunits, ankyrin(B), ankyrin(G), N-cadherin, or connexin-43. Together, these results suggest that beta1 is critical for normal cardiac excitability and loss of beta1 may be associated with a long QT phenotype.


Subject(s)
Heart Rate/genetics , Long QT Syndrome/genetics , Sodium Channels/deficiency , Animals , Brain/physiology , Electrocardiography , Heart/physiology , Heart Rate/physiology , Long QT Syndrome/physiopathology , Mice , Mice, Knockout , Muscle Cells/cytology , Muscle Cells/physiology , Patch-Clamp Techniques , Rats , Rats, Sprague-Dawley , Sodium Channels/physiology , Voltage-Gated Sodium Channel beta-1 Subunit
13.
Am J Physiol Heart Circ Physiol ; 293(6): H3558-67, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17890431

ABSTRACT

Previous studies have shown that cardiac inward rectifier potassium current (I(K1)) channels are heteromers of distinct Kir2 subunits and suggested that species- and tissue-dependent expression of these subunits may underlie variability of I(K1). In this study, we investigated the contribution of the slowly activating Kir2.3 subunit and free intracellular polyamines (PAs) to variability of I(K1) in the mouse heart. The kinetics of activation was measured in Kir2 concatemeric tetramers with known subunit stoichiometry. Inclusion of only one Kir2.3 subunit to a Kir2.1 channel led to an approximate threefold slowing of activation kinetics, with greater slowing on subsequent additions of Kir2.3 subunits. Activation kinetics of I(K1) in both ventricles and both atria was found to correspond to fast-activating Kir2.1/Kir2.2 channels, suggesting no major contribution of Kir2.3 subunits. In contrast, I(K1) displayed significant variation in both the current density and inward rectification, suggesting involvement of intracellular PAs. The total levels of PAs were similar across the mouse heart. Measurements of the free intracellular PAs in isolated myocytes, using transgenically expressed Kir2.1 channels as PA sensors, revealed "microheterogeneity" of I(K1) rectification as well as lower levels of free PAs in atrial myocytes compared with ventricular cells. These findings provide a quantitative explanation for the regional heterogeneity of I(K1).


Subject(s)
Myocytes, Cardiac/metabolism , Polyamines/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Potassium/metabolism , Animals , Cell Line , Green Fluorescent Proteins/metabolism , Heart Atria/metabolism , Heart Ventricles/metabolism , Humans , Kinetics , Membrane Potentials , Mice , Mice, Transgenic , Potassium Channels, Inwardly Rectifying/genetics , Protein Subunits/metabolism , Recombinant Fusion Proteins/metabolism , Transfection
14.
Basic Res Cardiol ; 102(5): 416-28, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17546530

ABSTRACT

The role of the cardiac current Ik1 in arrhythmogenesis remains highly controversal. To gain further insights into the mechanisms of IK1 involvement in cardiac excitability, we studied the susceptibility of transgenic mice with altered IK1 to arrhythmia during various pharmacological and physiological challenges. Arrhythmogenesis was studied in transgenic mice expressing either dominant negative Kir2.1-AAA or wild type Kir2.1 subunits in the heart, models of IK1 suppression (AAA-TG) and up-regulation (WT-TG), respectively. Under normal conditions, both anesthetized wild type (WT) and AAA-TG mice did not display any spontaneous arrhythmias. In contrast,WT-TG mice displayed numerous arrhythmias of various types. In isolated hearts, the threshold concentration for halothane-induced ventricular tachycardias (VT) was increased to 167% [corrected] in the AAA-TG and decreased to 54% [corrected] in WT-TG hearts when compared to WT hearts. The number of PVCs induced by AV node ablation combined with hypokalemia was reduced in AAA-TG hearts and increased in WT-TG mice. After AV node ablation AAA-TG hearts were more tolerant, and WT-TG less tolerant to isoproterenol- induced arrhythmias than WT hearts. Analysis of monophasic action potentials in isolated hearts shows a significant reduction in the dispersion of action potential repolarization in mice with suppressed IK1. The data strongly support the hypothesis that in the mouse heart upregulation of IK1 is proarrhythmic, and that under certain conditions IK1 blockade in cardiac myocytes may be a potentially useful antiarrhythmic strategy.


Subject(s)
Action Potentials/physiology , Arrhythmias, Cardiac/physiopathology , Potassium Channels, Inwardly Rectifying/genetics , Action Potentials/drug effects , Anesthetics, Inhalation/pharmacology , Animals , Arrhythmias, Cardiac/chemically induced , Arrhythmias, Cardiac/therapy , Cardiotonic Agents/pharmacology , Catheter Ablation , Electrocardiography , Female , Halothane/pharmacology , Hypokalemia/physiopathology , Isoproterenol/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Potassium Channels, Inwardly Rectifying/metabolism , Up-Regulation/physiology
15.
J Mol Cell Cardiol ; 43(1): 27-38, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17498734

ABSTRACT

It is established that prolonged hypoxia leads to activation of K(ATP) channels and action potential (AP) shortening, but the mechanisms behind the early phase of metabolic stress remain controversial. Under normal conditions IK1 channels are constitutively active while K(ATP) channels are closed. Therefore, early changes in IK1 may underlie early AP shortening. This hypothesis was tested using transgenic mice with suppressed IK1 (AAA-TG). In isolated AAA-TG hearts AP shortening was delayed by approximately 24 s compared to WT hearts. In WT ventricular myocytes, blocking oxidative phosphorylation with 1 mM cyanide (CN; 28 degrees C) led to a 29% decrease in APD90 within approximately 3-5 min. The effect of CN was reversed by application of 100 microM Ba2+, a selective blocker of IK1, but not by 10 microM glybenclamide, a selective blocker of KATP channels. Accordingly, voltage-clamp experiments revealed that both CN and true hypoxia lead to early activation of IK1. In AAA-TG myocytes, neither CN nor glybenclamide or Ba2+ had any effect on AP. Further experiments showed that buffering of intracellular Ca2+ with 20 mM BAPTA prevented IK1 activation by CN, although CN still caused a 54% increase in IK1 in a Ca2+ -free bath solution. Importantly, both (i) 20 microM ruthenium red, a selective inhibitor of SR Ca2+ -release, and (ii) depleting SR by application of 10 microM ryanodine+1 mM caffeine, abolished the activation of IK1 by CN. The above data strongly argue that in the mouse heart IK1, not KATP, channels are responsible for the early AP shortening during hypoxia.


Subject(s)
Myocytes, Cardiac/physiology , Potassium Channels, Inwardly Rectifying/metabolism , Action Potentials/genetics , Animals , Animals, Genetically Modified , Calcium/metabolism , Cell Hypoxia/genetics , Cells, Cultured , Mice , Organ Culture Techniques , Patch-Clamp Techniques , Potassium Channels, Inwardly Rectifying/genetics , Time Factors
16.
Am J Physiol Heart Circ Physiol ; 293(1): H836-45, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17449558

ABSTRACT

Sarcolemmal ATP-sensitive potassium (K(ATP)) channels are activated after pathological depletion of intracellular ATP, unlike their pancreatic beta-cell counterparts, which dynamically regulate membrane excitability in response to changes in blood glucose. We recently engineered a series of transgenic (TG) mice overexpressing an ATP-insensitive inward rectifying K(+) channel protein (Kir)6.2 mutant (Kir6.2[DeltaN30,K185Q]) or the accessory sulfonylurea receptor (SUR)2A (FLAG-SUR2A) or SUR1 (FLAG-SUR1) subunits of the K(ATP) channel, under transcriptional control of the alpha-myosin heavy chain promoter. In the present study, we generated double transgenic (DTG) animals overexpressing both Kir6.2[DeltaN30,K185Q] and FLAG-SUR1 or FLAG-SUR2A and examined the effects on cardiac excitability in vivo. No animals expressing both FLAG-SUR1 and Kir6.2[DeltaN30,K185Q] transgenes at a high level were obtained. DTG mice expressing one transgene at a high level and the other at a lower level are born, but they die prematurely. Electrocardiographic analysis of both anesthetized and conscious animals revealed a constellation of arrhythmias in DTG animals, but not in wild-type or single TG littermates. The proarrhythmic effect of the transgene combination is intrinsic to the myocardium, since it persists in isolated hearts. Importantly, this effect is specific for SUR1-expressing DTG animals: DTG animals expressing both Kir6.2[DeltaN30,K185Q] and FLAG-SUR2A at high levels exhibit neither impaired survival nor increased arrhythmia frequency, even with both subunits expressed at high levels. In demonstrating the profound arrhythmic consequences of K(ATP) channels comprised of SUR1 and Kir6.2[DeltaN30,K185Q] in the myocardium specifically, the results highlight the critical differential activation of SUR1 versus SUR2A, and indicate that expression of hyperactive K(ATP) in the heart is likely to be proarrhythmic.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Arrhythmias, Cardiac/genetics , Arrhythmias, Cardiac/metabolism , Multidrug Resistance-Associated Proteins/metabolism , Myocardium/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , ATP-Binding Cassette Transporters/genetics , Animals , Female , Genetic Predisposition to Disease/genetics , Male , Mice , Mice, Transgenic , Multidrug Resistance-Associated Proteins/genetics , Potassium Channels, Inwardly Rectifying/genetics , Receptors, Drug , Sulfonylurea Receptors , Survival Analysis , Survival Rate , Up-Regulation
17.
Am J Physiol Heart Circ Physiol ; 292(5): H2532-9, 2007 May.
Article in English | MEDLINE | ID: mdl-17277016

ABSTRACT

G protein-coupled receptors play a pivotal role in regulating cardiac automaticity. Their function is controlled by regulator of G protein signaling (RGS) proteins acting as GTPase-activating proteins for Galpha subunits to suppress Galpha(i) and Galpha(q) signaling. Using knock-in mice in which Galpha(i2)-RGS binding and negative regulation are disrupted by a genomic Galpha(i2)G184S (GS) point mutation, we recently (Fu Y, Huang X, Zhong H, Mortensen RM, D'Alecy LG, Neubig RR. Circ Res 98: 659-666, 2006) showed that endogenous RGS proteins suppress muscarinic receptor-mediated bradycardia. To determine whether this was due to direct regulation of cardiac pacemakers or to alterations in the central nervous system or vascular responses, we examined isolated, perfused hearts. Isoproterenol-stimulated beating rates of heterozygote (+/GS) and homozygote (GS/GS) hearts were significantly more sensitive to inhibition by carbachol than were those of wild type (+/+). Even greater effects were seen in the absence of isoproterenol; the potency of muscarinic-mediated bradycardia was enhanced fivefold in GS/GS and twofold in +/GS hearts compared with +/+. A(1)-adenosine receptor-mediated bradycardia was unaffected. In addition to effects on the sinoatrial node, +/GS and GS/GS hearts show significantly increased carbachol-induced third-degree atrioventricular (AV) block. Atrial pacing studies demonstrated an increased PR interval and AV effective refractory period in GS/GS hearts compared with +/+. Thus loss of the inhibitory action of endogenous RGS proteins on Galpha(i2) potentiates muscarinic inhibition of cardiac automaticity and conduction. The severe carbachol-induced sinus bradycardia in Galpha(i2)G184S mice suggests a possible role for alterations of Galpha(i2) or RGS proteins in sick sinus syndrome and pathological AV block.


Subject(s)
Atrioventricular Node/physiopathology , Heart Block/physiopathology , Heart Rate , RGS Proteins/metabolism , Sick Sinus Syndrome/physiopathology , Sinoatrial Node/physiopathology , Animals , In Vitro Techniques , Mice , Mice, Knockout
18.
J Physiol ; 578(Pt 1): 315-26, 2007 Jan 01.
Article in English | MEDLINE | ID: mdl-17095564

ABSTRACT

Previous studies have suggested an important role for the inward rectifier K+ current (I K1) in stabilizing rotors responsible for ventricular tachycardia (VT) and fibrillation (VF). To test this hypothesis, we used a line of transgenic mice (TG) overexpressing Kir 2.1-green fluorescent protein (GFP) fusion protein in a cardiac-specific manner. Optical mapping of the epicardial surface in ventricles showed that the Langendorff-perfused TG hearts were able to sustain stable VT/VF for 350 +/- 1181 s at a very high dominant frequency (DF) of 44.6 +/- 4.3 Hz. In contrast, tachyarrhythmias in wild-type hearts (WT) were short-lived (3 +/- 9 s), and the DF was 26.3 +/- 5.2 Hz. The stable, high frequency, reentrant activity in TG hearts slowed down, and eventually terminated in the presence of 10 mum Ba2+, suggesting an important role for I K1. Moreover, by increasing I K1 density in a two-dimensional computer model having realistic mouse ionic and action potential properties, a highly stable, fast rotor (approximately 45 Hz) could be induced. Simulations suggested that the TG hearts allowed such a fast and stable rotor because of both greater outward conductance at the core and shortened action potential duration in the core vicinity, as well as increased excitability, in part due to faster recovery of Na+ current. The latter resulted in a larger rate of increase in the local conduction velocity as a function of the distance from the core in TG compared to WT hearts, in both simulations and experiments. Finally, simulations showed that rotor frequencies were more sensitive to changes (doubling) in I K1, compared to other K+ currents. In combination, these results provide the first direct evidence that I K1 up-regulation in the mouse heart is a substrate for stable and very fast rotors.


Subject(s)
Heart/physiology , Potassium Channels, Inwardly Rectifying/biosynthesis , Potassium Channels, Inwardly Rectifying/physiology , Animals , Arrhythmias, Cardiac/drug therapy , Arrhythmias, Cardiac/physiopathology , Atrial Fibrillation/physiopathology , Atrial Flutter/physiopathology , Cardiomegaly/physiopathology , Computer Simulation , Death, Sudden , Electrocardiography , Heart Block/physiopathology , Heart Conduction System/physiology , Heart Rate/physiology , In Vitro Techniques , Mice , Mice, Transgenic , Potassium Channel Blockers/pharmacology , Potassium Channels, Inwardly Rectifying/genetics , Refractory Period, Electrophysiological/genetics , Refractory Period, Electrophysiological/physiology , Up-Regulation/physiology
19.
J Physiol ; 571(Pt 2): 287-302, 2006 Mar 01.
Article in English | MEDLINE | ID: mdl-16373386

ABSTRACT

Recent studies have shown that Kir2 channels display differential sensitivity to intracellular polyamines, and have raised a number of questions about several properties of inward rectification important to the understanding of their physiological roles. In this study, we have carried out a detailed characterization of steady-state and kinetic properties of block of Kir2.1-3 channels by spermine. High-resolution recordings from outside-out patches showed that in all Kir2 channels current-voltage relationships display a 'crossover' effect upon change in extracellular K+. Experiments at different concentrations of spermine allowed for the characterization of two distinct shallow components of rectification, with the voltages for half-block negative (V1(1/2)) and positive (V2(1/2)) to the voltage of half-block for the major steep component of rectification (V0(1/2)). While V1(1/2) and V2(1/2) voltages differ significantly between Kir2 channels, they were coupled to each other according to the equation V1(1/2)-V2(1/2) = constant, strongly suggesting that similar structures may underlie both components. In Kir2.3 channels, the V2(1/2) was approximately 50 mV positive to V0(1/2), leading to a pattern of outward currents distinct from that of Kir2.1 and Kir2.2 channels. The effective valency of spermine block (Z0) was highest in Kir2.2 channels while the valencies in Kir2.1 and Kir2.3 channels were not significantly different. The voltage dependence of spermine unblock was similar in all Kir2 channels, but the rates of unblock were approximately 7-fold and approximately 16-fold slower in Kir2.3 channels than those in Kir2.1 and Kir2.2 when measured at high and physiological extracellular K+, respectively. In all Kir2 channels, the instantaneous phase of activation was present. The instantaneous phase was difficult to resolve at high extracellular K+ but it became evident and accounted for nearly 30-50% of the total current when recorded at physiological extracellular K+. In conclusion, the data are consistent with the universal mechanism of rectification in Kir2 channels, but also point to significant, and physiologically important, quantitative differences between Kir2 isoforms.


Subject(s)
Polyamines/metabolism , Potassium Channels, Inwardly Rectifying/chemistry , Amino Acid Sequence , Dose-Response Relationship, Drug , Humans , Membrane Potentials/drug effects , Membrane Potentials/physiology , Molecular Sequence Data , Mutation , Polyamines/antagonists & inhibitors , Potassium/metabolism , Potassium/pharmacology , Potassium Channels, Inwardly Rectifying/antagonists & inhibitors , Potassium Channels, Inwardly Rectifying/genetics , Potassium Channels, Inwardly Rectifying/physiology , Protein Structure, Secondary , Sequence Homology , Spermine/pharmacology , Transfection
20.
J Mol Cell Cardiol ; 39(4): 647-56, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16099470

ABSTRACT

The lack of pathological consequences of cardiac ATP-sensitive potassium channel (K(ATP)) channel gene manipulation is in stark contrast to the effect of similar perturbations in the pancreatic beta-cell. Because the pancreatic and cardiac channel share the same pore-forming subunit (Kir6.2), the different effects of genetic manipulation likely reflect, at least in part, the tissue-specific expression of the regulatory subunit (SUR1 in pancreas vs. SUR2A in heart) of the bipartite channel complex. To examine this, we have generated transgenic (TG) mice that overexpress epitope-tagged SUR1 or SUR2A under the transcriptional control of the alpha-myosin heavy chain promoter. Western blot and real time RT-PCR analysis confirm transgene expression in the heart, and variable levels of SUR1 RNA and protein, in 16 viable founder lines. Surprisingly, activation of channels by either pharmacological agents (diazoxide and pinacidil) or metabolic inhibitors (oligomycin and 2-deoxyglucose) reveals a suppression of total K(ATP) conductance in high expressing TG mice. Moreover, K(ATP) channel activity was significantly reduced in excised cardiac patches from TG myocytes that overexpress either SUR1 or SUR2A. Using a recombinant cell system, we show that overexpression of either SUR1 or Kir6.2 suppresses the functional expression of K(ATP) from optimized dimeric SUR1-Kir6.2. Thus, the graded effect of SUR1 expression in the intact heart appears to demonstrate an in vivo requirement for 1:1 expression ratio of Kir6.2 and SURx.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Myocardium/metabolism , Myocytes, Cardiac/physiology , Potassium Channels, Inwardly Rectifying/metabolism , Potassium Channels/metabolism , Receptors, Drug/metabolism , ATP-Binding Cassette Transporters/agonists , ATP-Binding Cassette Transporters/genetics , Animals , Diazoxide/pharmacology , Mice , Mice, Transgenic , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Myosin Heavy Chains/genetics , Potassium Channels/agonists , Potassium Channels/genetics , Potassium Channels, Inwardly Rectifying/agonists , Potassium Channels, Inwardly Rectifying/drug effects , Potassium Channels, Inwardly Rectifying/genetics , Promoter Regions, Genetic/genetics , Receptors, Drug/agonists , Receptors, Drug/genetics , Sarcolemma/metabolism , Sulfonylurea Receptors , Transcriptional Activation , Ventricular Myosins/genetics
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