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1.
Am J Physiol Heart Circ Physiol ; 318(6): H1357-H1370, 2020 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-32196358

RESUMEN

Synapse-associated protein 97 (SAP97) is a scaffolding protein crucial for the functional expression of several cardiac ion channels and therefore proper cardiac excitability. Alterations in the functional expression of SAP97 can modify the ionic currents underlying the cardiac action potential and consequently confer susceptibility for arrhythmogenesis. In this study, we generated a murine model for inducible, cardiac-targeted Sap97 ablation to investigate arrhythmia susceptibility and the underlying molecular mechanisms. Furthermore, we sought to identify human SAP97 (DLG1) variants that were associated with inherited arrhythmogenic disease. The murine model of cardiac-specific Sap97 ablation demonstrated several ECG abnormalities, pronounced action potential prolongation subject to high incidence of arrhythmogenic afterdepolarizations and notable alterations in the activity of the main cardiac ion channels. However, no DLG1 mutations were found in 40 unrelated cases of genetically elusive long QT syndrome (LQTS). Instead, we provide the first evidence implicating a gain of function in human DLG1 mutation resulting in an increase in Kv4.3 current (Ito) as a novel, potentially pathogenic substrate for Brugada syndrome (BrS). In conclusion, DLG1 joins a growing list of genes encoding ion channel interacting proteins (ChIPs) identified as potential channelopathy-susceptibility genes because of their ability to regulate the trafficking, targeting, and modulation of ion channels that are critical for the generation and propagation of the cardiac electrical impulse. Dysfunction in these critical components of cardiac excitability can potentially result in fatal cardiac disease.NEW & NOTEWORTHY The gene encoding SAP97 (DLG1) joins a growing list of genes encoding ion channel-interacting proteins (ChIPs) identified as potential channelopathy-susceptibility genes because of their ability to regulate the trafficking, targeting, and modulation of ion channels that are critical for the generation and propagation of the cardiac electrical impulse. In this study we provide the first data supporting DLG1-encoded SAP97's candidacy as a minor Brugada syndrome susceptibility gene.


Asunto(s)
Arritmias Cardíacas/metabolismo , Homólogo 1 de la Proteína Discs Large/metabolismo , Corazón/fisiopatología , Miocardio/metabolismo , Animales , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatología , Homólogo 1 de la Proteína Discs Large/genética , Humanos , Ratones , Ratones Noqueados , Miocitos Cardíacos/metabolismo
2.
Heart Fail Clin ; 12(2): 157-66, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26968662

RESUMEN

Atrial fibrillation (AF) is by far the most common sustained tachyarrhythmia, affecting 1% to 2% of the general population. AF prevalence and the total annual cost for treatment are alarming, emphasizing the need for an urgent attention to the problem. Thus, having up-to-date information on AF risk factors and appreciating how they promote maintenance of AF maintenance are essential. This article presents a simplified examination of AF risk factors, including emerging genetic risks.

3.
Proc Natl Acad Sci U S A ; 109(31): E2134-43, 2012 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-22509027

RESUMEN

The cardiac electrical impulse depends on an orchestrated interplay of transmembrane ionic currents in myocardial cells. Two critical ionic current mechanisms are the inwardly rectifying potassium current (I(K1)), which is important for maintenance of the cell resting membrane potential, and the sodium current (I(Na)), which provides a rapid depolarizing current during the upstroke of the action potential. By controlling the resting membrane potential, I(K1) modifies sodium channel availability and therefore, cell excitability, action potential duration, and velocity of impulse propagation. Additionally, I(K1)-I(Na) interactions are key determinants of electrical rotor frequency responsible for abnormal, often lethal, cardiac reentrant activity. Here, we have used a multidisciplinary approach based on molecular and biochemical techniques, acute gene transfer or silencing, and electrophysiology to show that I(K1)-I(Na) interactions involve a reciprocal modulation of expression of their respective channel proteins (Kir2.1 and Na(V)1.5) within a macromolecular complex. Thus, an increase in functional expression of one channel reciprocally modulates the other to enhance cardiac excitability. The modulation is model-independent; it is demonstrable in myocytes isolated from mouse and rat hearts and with transgenic and adenoviral-mediated overexpression/silencing. We also show that the post synaptic density, discs large, and zonula occludens-1 (PDZ) domain protein SAP97 is a component of this macromolecular complex. We show that the interplay between Na(v)1.5 and Kir2.1 has electrophysiological consequences on the myocardium and that SAP97 may affect the integrity of this complex or the nature of Na(v)1.5-Kir2.1 interactions. The reciprocal modulation between Na(v)1.5 and Kir2.1 and the respective ionic currents should be important in the ability of the heart to undergo self-sustaining cardiac rhythm disturbances.


Asunto(s)
Potenciales de Acción , Arritmias Cardíacas/mortalidad , Regulación de la Expresión Génica , Potenciales de la Membrana , Proteínas Musculares/biosíntesis , Miocitos Cardíacos/metabolismo , Canales de Potasio de Rectificación Interna/biosíntesis , Canales de Sodio/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatología , Homólogo 1 de la Proteína Discs Large , Silenciador del Gen , Guanilato-Quinasas/genética , Guanilato-Quinasas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Proteínas Musculares/genética , Miocitos Cardíacos/patología , Canal de Sodio Activado por Voltaje NAV1.5 , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Canales de Potasio de Rectificación Interna/genética , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Canales de Sodio/genética , Proteína de la Zonula Occludens-1
4.
Circ Res ; 111(7): 842-53, 2012 Sep 14.
Artículo en Inglés | MEDLINE | ID: mdl-22843785

RESUMEN

RATIONALE: Kv1.5 (KCNA5) is expressed in the heart, where it underlies the I(Kur) current that controls atrial repolarization, and in the pulmonary vasculature, where it regulates vessel contractility in response to changes in oxygen tension. Atrial fibrillation and hypoxic pulmonary hypertension are characterized by downregulation of Kv1.5 protein expression, as well as with oxidative stress. Formation of sulfenic acid on cysteine residues of proteins is an important, dynamic mechanism for protein regulation under oxidative stress. Kv1.5 is widely reported to be redox-sensitive, and the channel possesses 6 potentially redox-sensitive intracellular cysteines. We therefore hypothesized that sulfenic acid modification of the channel itself may regulate Kv1.5 in response to oxidative stress. OBJECTIVE: To investigate how oxidative stress, via redox-sensitive modification of the channel with sulfenic acid, regulates trafficking and expression of Kv1.5. METHODS AND RESULTS: Labeling studies with the sulfenic acid-specific probe DAz and horseradish peroxidase-streptavidin Western blotting demonstrated a global increase in sulfenic acid-modified proteins in human patients with atrial fibrillation, as well as sulfenic acid modification to Kv1.5 in the heart. Further studies showed that Kv1.5 is modified with sulfenic acid on a single COOH-terminal cysteine (C581), and the level of sulfenic acid increases in response to oxidant exposure. Using live-cell immunofluorescence and whole-cell voltage-clamping, we found that modification of this cysteine is necessary and sufficient to reduce channel surface expression, promote its internalization, and block channel recycling back to the cell surface. Moreover, Western blotting demonstrated that sulfenic acid modification is a trigger for channel degradation under prolonged oxidative stress. CONCLUSIONS: Sulfenic acid modification to proteins, which is elevated in diseased human heart, regulates Kv1.5 channel surface expression and stability under oxidative stress and diverts channel from a recycling pathway to degradation. This provides a molecular mechanism linking oxidative stress and downregulation of channel expression observed in cardiovascular diseases.


Asunto(s)
Fibrilación Atrial/metabolismo , Canal de Potasio Kv1.5/química , Canal de Potasio Kv1.5/metabolismo , Miocardio/metabolismo , Ácidos Sulfénicos/metabolismo , Secuencia de Aminoácidos , Animales , Fibrilación Atrial/patología , Estudios de Casos y Controles , Línea Celular , Células Cultivadas , Humanos , Ratones , Modelos Animales , Datos de Secuencia Molecular , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Oxidación-Reducción , Estrés Oxidativo/fisiología , Ratas , Especies Reactivas de Oxígeno , Transducción de Señal/fisiología
5.
bioRxiv ; 2024 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-39149279

RESUMEN

Obesity is a major risk factor for atrial fibrillation (AF) the most common serious cardiac arrhythmia, but the molecular mechanisms underlying diet-induced AF remain unclear. In this study, we subjected mice to a chronic high-fat diet and acute sympathetic activation ('two-hit' model) to study the mechanisms by which diet-induced obesity promotes AF. Surface electrocardiography revealed that diet-induced obesity and sympathetic activation synergize during intracardiac tachypacing to induce AF. At the cellular level, diet-induced obesity and acute adrenergic stimulation facilitate the formation of delayed afterdepolarizations in atrial myocytes, implicating altered Ca2+ dynamics as the underlying cause of AF. We found that diet-induced obesity does not alter the expression of major Ca2+-handling proteins in atria, including the sarcoplasmic reticulum Ca2+-ATPase (SERCA), a major component of beat-to-beat Ca2+ cycling in the heart. Paradoxically, obesity reduces phospholamban phosphorylation, suggesting decreased SERCA activity, yet atrial myocytes from obese mice showed a significantly increased Ca2+ transient amplitude and SERCA-mediated Ca2+ uptake. Adrenergic stimulation further increases the Ca2+ transient amplitude but does not affect Ca2+ reuptake in atrial myocytes from obese mice. Transcriptomics analysis showed that a high-fat diet prompts upregulation of neuronatin, a protein that has been implicated in obesity and is known to stimulate SERCA activity. We propose a mechanism in which obesity primes SERCA for paradoxical activation, and adrenergic stimulation facilitates AF conversion through a Ca2+-induced Ca2+ release gain in atrial myocytes. Overall, this study links obesity, altered Ca2+ signaling, and AF, and targeting this mechanism may prove effective for treating obesity-induced AF.

8.
J Biol Chem ; 285(36): 28000-9, 2010 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-20530486

RESUMEN

Synapse-associated protein-97 (SAP97) is a membrane-associated guanylate kinase scaffolding protein expressed in cardiomyocytes. SAP97 has been shown to associate and modulate voltage-gated potassium (Kv) channel function. In contrast to Kv channels, little information is available on interactions involving SAP97 and inward rectifier potassium (Kir2.x) channels that underlie the classical inward rectifier current, I(K1). To investigate the functional effects of silencing SAP97 on I(K1) in adult rat ventricular myocytes, SAP97 was silenced using an adenoviral short hairpin RNA vector. Western blot analysis showed that SAP97 was silenced by approximately 85% on day 3 post-infection. Immunostaining showed that Kir2.1 and Kir2.2 co-localize with SAP97. Co-immunoprecipitation (co-IP) results demonstrated that Kir2.x channels associate with SAP97. Voltage clamp experiments showed that silencing SAP97 reduced I(K1) whole cell density by approximately 55%. I(K1) density at -100 mV was -1.45 +/- 0.15 pA/picofarads (n = 6) in SAP97-silenced cells as compared with -3.03 +/- 0.37 pA/picofarads (n = 5) in control cells. Unitary conductance properties of I(K1) were unaffected by SAP97 silencing. The major mechanism for the reduction of I(K1) density appears to be a decrease in Kir2.x channel abundance. Furthermore, SAP97 silencing impaired I(K1) regulation by beta(1)-adrenergic receptor (beta1-AR) stimulation. In control, isoproterenol reduced I(K1) amplitude by approximately 75%, an effect that was blunted following SAP97 silencing. Our co-IP data show that beta1-AR associates with SAP97 and Kir2.1 and also that Kir2.1 co-IPs with protein kinase A and beta1-AR. SAP97 immunolocalizes with protein kinase A and beta1-AR in the cardiac myocytes. Our results suggest that in cardiac myocytes SAP97 regulates surface expression of channels underlying I(K1), as well as assembles a signaling complex involved in beta1-AR regulation of I(K1).


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Conductividad Eléctrica , Proteínas de la Membrana/metabolismo , Miocardio/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Inmunoprecipitación , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Células Musculares/metabolismo , Transporte de Proteínas , Ratas , Receptores Adrenérgicos beta 1/metabolismo
10.
J Mol Cell Cardiol ; 48(1): 45-54, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19703462

RESUMEN

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.


Asunto(s)
Corazón/fisiología , Miocardio/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Potenciales de Acción/genética , Potenciales de Acción/fisiología , Animales , Corazón/fisiopatología , Humanos , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Modelos Biológicos , Miocardio/patología , Canales de Potasio de Rectificación Interna/genética
11.
Biophys J ; 96(7): 2961-76, 2009 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-19348777

RESUMEN

The mechanisms controlling the rotation frequency of functional reentry in ventricular fibrillation (VF) are poorly understood. It has been previously shown that Ba2+ at concentrations up to 50 mumol/L slows the rotation frequency in the intact guinea pig (GP) heart, suggesting a role of the inward rectifier current (I(K1)) in the mechanism governing the VF response to Ba2+. Given that other biological (e.g., sinoatrial node) and artificial systems display phase-locking behavior, we hypothesized that the mechanism for controlling the rotation frequency of a rotor by I(K1) blockade is phase-driven, i.e., the phase shift between transmembrane current and voltage remains constant at varying levels of I(K1) blockade. We measured whole-cell admittance in isolated GP myocytes and in transfected human embryonic kidney (HEK) cells stably expressing Kir 2.1 and 2.3 channels. The admittance phase, i.e., the phase difference between current and voltage, was plotted versus the frequency in control conditions and at 10 or 50 micromol/L Ba2+ (in GP heart cells) or 1 mM Ba2+ (in HEK cells). The horizontal distance between plots was called the "frequency shift in a single cell" and analyzed. The frequency shift in a single cell was -14.14 +/- 5.71 Hz (n = 14) at 10 microM Ba2+ and -18.51 +/- 4.00 Hz (n = 10) at 50 microM Ba2+, p < 0.05. The values perfectly matched the Ba2+-induced reduction of VF frequency observed previously in GP heart. A similar relationship was found in the computer simulations. The phase of Ba2+-sensitive admittance in GP cells was -2.65 +/- 0.32 rad at 10 Hz and -2.79 +/- 0.26 rad at 30 Hz. In HEK cells, the phase of Ba2+-sensitive admittance was 3.09 +/- 0.03 rad at 10 Hz and 3.00 +/- 0.17 rad at 30 Hz. We have developed a biological single-cell model of rotation-frequency control. The results show that although rotation frequency changes as a result of I(K1) blockade, the phase difference between transmembrane current and transmembrane voltage remains constant, enabling us to quantitatively predict the change of VF frequency resulting from I(K1) blockade, based on single-cell measurement.


Asunto(s)
Modelos Biológicos , Fibrilación Ventricular/patología , Animales , Bario/farmacología , Línea Celular , Fenómenos Fisiológicos Celulares/efectos de los fármacos , Simulación por Computador , Conductividad Eléctrica , Cobayas , Humanos , Células Musculares/efectos de los fármacos , Células Musculares/metabolismo , Células Musculares/patología , Sensibilidad y Especificidad , Fibrilación Ventricular/metabolismo
12.
Am J Physiol Heart Circ Physiol ; 297(4): H1387-97, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19633205

RESUMEN

We examined the impact of coexpressing the inwardly rectifying potassium channel, Kir2.3, with the scaffolding protein, synapse-associated protein (SAP) 97, and determined that coexpression of these proteins caused an approximately twofold increase in current density. A combination of techniques was used to determine if the SAP97-induced increase in Kir2.3 whole cell currents resulted from changes in the number of channels in the cell membrane, unitary channel conductance, or channel open probability. In the absence of SAP97, Kir2.3 was found predominantly in a cytoplasmic, vesicular compartment with relatively little Kir2.3 localized to the plasma membrane. The introduction of SAP97 caused a redistribution of Kir2.3, leading to prominent colocalization of Kir2.3 and SAP97 and a modest increase in cell surface Kir2.3. The median Kir2.3 single channel conductance in the absence of SAP97 was approximately 13 pS, whereas coexpression of SAP97 led to a wide distribution of channel events with three distinct peaks centered at 16, 29, and 42 pS. These changes occurred without altering channel open probability, current rectification properties, or pH sensitivity. Thus association of Kir2.3 with SAP97 in HEK293 cells increased channel cell surface expression and unitary channel conductance. However, changes in single channel conductance play the major role in determining whole cell currents in this model system. We further suggest that the SAP97 effect results from SAP97 binding to the Kir2.3 COOH-terminal domain and altering channel conformation.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Activación del Canal Iónico , Proteínas de la Membrana/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Sitios de Unión , Línea Celular , Membrana Celular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Cobayas , Atrios Cardíacos/metabolismo , Humanos , Potenciales de la Membrana , Proteínas de la Membrana/genética , Miocardio/metabolismo , Canales de Potasio de Rectificación Interna/genética , Conformación Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Ratas , Ovinos , Transfección
13.
Circ Res ; 101(10): 1039-48, 2007 Nov 09.
Artículo en Inglés | MEDLINE | ID: mdl-17872467

RESUMEN

Catecholaminergic polymorphic ventricular tachycardia (VT) is a lethal familial disease characterized by bidirectional VT, polymorphic VT, and ventricular fibrillation. Catecholaminergic polymorphic VT is caused by enhanced Ca2+ release through defective ryanodine receptor (RyR2) channels. We used epicardial and endocardial optical mapping, chemical subendocardial ablation with Lugol's solution, and patch clamping in a knockin (RyR2/RyR2(R4496C)) mouse model to investigate the arrhythmogenic mechanisms in catecholaminergic polymorphic VT. In isolated hearts, spontaneous ventricular arrhythmias occurred in 54% of 13 RyR2/RyR2(R4496C) and in 9% of 11 wild-type (P=0.03) littermates perfused with Ca2+and isoproterenol; 66% of 12 RyR2/RyR2(R4496C) and 20% of 10 wild-type hearts perfused with caffeine and epinephrine showed arrhythmias (P=0.04). Epicardial mapping showed that monomorphic VT, bidirectional VT, and polymorphic VT manifested as concentric epicardial breakthrough patterns, suggesting a focal origin in the His-Purkinje networks of either or both ventricles. Monomorphic VT was clearly unifocal, whereas bidirectional VT was bifocal. Polymorphic VT was initially multifocal but eventually became reentrant and degenerated into ventricular fibrillation. Endocardial mapping confirmed the Purkinje fiber origin of the focal arrhythmias. Chemical ablation of the right ventricular endocardial cavity with Lugol's solution induced complete right bundle branch block and converted the bidirectional VT into monomorphic VT in 4 anesthetized RyR2/RyR2(R4496C) mice. Under current clamp, single Purkinje cells from RyR2/RyR2(R4496C) mouse hearts generated delayed afterdepolarization-induced triggered activity at lower frequencies and level of adrenergic stimulation than wild-type. Overall, the data demonstrate that the His-Purkinje system is an important source of focal arrhythmias in catecholaminergic polymorphic VT.


Asunto(s)
Fascículo Atrioventricular/fisiología , Ramos Subendocárdicos/fisiología , Canal Liberador de Calcio Receptor de Rianodina/genética , Taquicardia Ventricular/genética , Taquicardia Ventricular/fisiopatología , Potenciales de Acción/fisiología , Animales , Calcio/metabolismo , Catecolaminas/fisiología , Muerte Súbita Cardíaca , Modelos Animales de Enfermedad , Endocardio/fisiología , Femenino , Masculino , Ratones , Ratones Mutantes , Técnicas de Placa-Clamp , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Taquicardia Ventricular/etiología
14.
Front Physiol ; 9: 2, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29403390

RESUMEN

Anatomical evidence in several species shows highly heterogeneous fat distribution in the atrial and ventricular myocardium. Atrial appendages have fat deposits, and more so on the posterior left atrium. Although such fat distributions are considered normal, fatty infiltration is regarded arrhythmogenic, and various cardiac pathophysiological conditions show excess myocardial fat deposits, especially in the epicardium. Hypotheses have been presented for the physiological and pathophysiological roles of epicardial fat, however this issue is poorly understood. Therefore, this mini-review will focus on epicardial fat distribution and the (patho)-physiological implications of this distribution. Potential molecular mechanisms that may drive structural and electrical myocardial remodeling attendant to fatty infiltration of the heart are also reviewed.

15.
Circ Arrhythm Electrophysiol ; 11(3): e005659, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29540372

RESUMEN

BACKGROUND: The mechanisms underlying spontaneous atrial fibrillation (AF) associated with atrial ischemia/infarction are incompletely elucidated. Here, we investigate the mechanisms underlying spontaneous AF in an ovine model of left atrial myocardial infarction (LAMI). METHODS AND RESULTS: LAMI was created by ligating the atrial branch of the left anterior descending coronary artery. ECG loop recorders were implanted to monitor AF episodes. In 7 sheep, dantrolene-a ryanodine receptor blocker-was administered in vivo during the 8-day observation period (LAMI-D, 2.5 mg/kg, IV, BID). LAMI animals experienced numerous spontaneous AF episodes during the 8-day monitoring period that were suppressed by dantrolene (LAMI, 26.1±5.1; sham, 4.3±1.1; LAMI-D, 2.8±0.8; mean±SEM episodes per sheep, P<0.01). Optical mapping showed spontaneous focal discharges (SFDs) originating from the ischemic/normal-zone border. SFDs were calcium driven, rate dependent, and enhanced by isoproterenol (0.03 µmol/L, from 210±87 to 3816±1450, SFDs per sheep) but suppressed by dantrolene (to 55.8±32.8, SFDs per sheep, mean±SEM). SFDs initiated AF-maintaining reentrant rotors anchored by marked conduction delays at the ischemic/normal-zone border. NOS1 (NO synthase-1) protein expression decreased in ischemic zone myocytes, whereas NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) oxidase and xanthine oxidase enzyme activities and reactive oxygen species (DCF [6-carboxy-2',7'-dichlorodihydrofluorescein diacetate]-fluorescence) increased. CaM (calmodulin) aberrantly increased [3H]ryanodine binding to cardiac RyR2 (ryanodine receptors) in the ischemic zone. Dantrolene restored the physiological binding of CaM to RyR2. CONCLUSIONS: Atrial ischemia causes spontaneous AF episodes in sheep, caused by SFDs that initiate reentry. Nitroso-redox imbalance in the ischemic zone is associated with intense reactive oxygen species production and altered RyR2 responses to CaM. Dantrolene administration normalizes the CaM response, prevents LAMI-related SFDs, and AF initiation. These findings provide novel insights into the mechanisms underlying ischemia-related atrial arrhythmias.


Asunto(s)
Fibrilación Atrial/complicaciones , Dantroleno/farmacología , Isquemia Miocárdica/etiología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Fibrilación Atrial/metabolismo , Fibrilación Atrial/fisiopatología , Fibrilación Atrial/terapia , Western Blotting , Señalización del Calcio , Modelos Animales de Enfermedad , Atrios Cardíacos , Masculino , Relajantes Musculares Centrales/farmacología , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatología , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Ovinos
16.
Heart Rhythm ; 4(4): 487-96, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17399639

RESUMEN

BACKGROUND: Data on pH regulation of the cardiac potassium current I(K1) suggest species-dependent differences in the molecular composition of the underlying Kir2 channel proteins. OBJECTIVE: The purpose of this study was to test the hypothesis that the presence of the Kir2.3 isoform in heterotetrameric channels modifies channel sensitivity to pH. METHODS: Voltage clamp was performed on HEK293 cells stably expressing guinea pig Kir2.1 and/or Kir2.3 isoforms and on sheep cardiac ventricular myocytes at varying extracellular pH (pH(o)) and in the presence of CO(2) to determine the sensitivity of macroscopic currents to pH. Single-channel activity was recorded from the HEK293 stables to determine the mechanisms of the changes in whole cell current. RESULTS: Biophysical characteristics of whole-cell and single-channel currents in Kir2.1/Kir2.3 double stables displayed properties attributable to isoform heteromerization. Whole-cell Kir2.1/Kir2.3 currents rectified in a manner reminiscent of Kir2.1 but were significantly inhibited by extracellular acidification in the physiologic range (pK(a) approximately 7.4). Whole-cell currents were more sensitive to a combined extracellular and intracellular acidification produced by CO(2). At pH(o) = 6.0, unitary conductances of heteromeric channels were reduced. Ovine cardiac ventricular cell I(K1) was pH(o) and CO(2) sensitive, consistent with the expression of Kir2.1 and Kir2.3 in this species. CONCLUSION: Kir2.1 and Kir2.3 isoforms form heteromeric channels in HEK293. The presence of Kir2.3 subunit(s) in heteromeric channels confers pH sensitivity to the channels. The single and double stable cells presented in this study are useful models for studying physiologic regulation of heteromeric Kir2 channels in mammalian cells.


Asunto(s)
Miocitos Cardíacos/fisiología , Canales de Potasio de Rectificación Interna/fisiología , Análisis de Varianza , Animales , Western Blotting , Línea Celular , Electrofisiología , Cobayas , Ventrículos Cardíacos/citología , Concentración de Iones de Hidrógeno , Modelos Animales , Oocitos/fisiología , Técnicas de Placa-Clamp , Isoformas de Proteínas , Proyectos de Investigación , Ovinos , Xenopus
17.
Circ Res ; 96(7): 800-7, 2005 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-15761194

RESUMEN

Short QT syndrome (SQTS) leads to an abbreviated QTc interval and predisposes patients to life-threatening arrhythmias. To date, two forms of the disease have been identified: SQT1, caused by a gain of function substitution in the HERG (I(Kr)) channel, and SQT2, caused by a gain of function substitution in the KvLQT1 (I(Ks)) channel. Here we identify a new variant, "SQT3", which has a unique ECG phenotype characterized by asymmetrical T waves, and a defect in the gene coding for the inwardly rectifying Kir2.1 (I(K1)) channel. The affected members of a single family had a G514A substitution in the KCNJ2 gene that resulted in a change from aspartic acid to asparagine at position 172 (D172N). Whole-cell patch-clamp studies of the heterologously expressed human D172N channel demonstrated a larger outward I(K1) than the wild-type (P<0.05) at potentials between -75 mV and -45 mV, with the peak current being shifted in the former with respect to the latter (WT, -75 mV; D172N, -65 mV). Coexpression of WT and mutant channels to mimic the heterozygous condition of the proband yielded an outward current that was intermediate between WT and D172N. In computer simulations using a human ventricular myocyte model the increased outward I(K1) greatly accelerated the final phase of repolarization, and shortened the action potential duration. Hence, unlike the known mutations in the two other SQTS forms (N588K in HERG and V307L in KvLQT1), simulations using the D172N and WT/D172N mutations fully accounted for the ECG phenotype of tall and asymmetrically shaped T waves. Although we were unable to test for inducibility of arrhythmia susceptibility due to lack of patients' consent, our computer simulations predict a steeper steady-state restitution curve for the D172N and WT/D172N mutation, compared with WT or to HERG or KvLQT1 mutations, which may predispose SQT3 patients to a greater risk of reentrant arrhythmias.


Asunto(s)
Electrocardiografía , Mutación , Canales de Potasio de Rectificación Interna/genética , Taquicardia/etiología , Potenciales de Acción , Animales , Células CHO , Preescolar , Cricetinae , Femenino , Humanos , Canales de Potasio de Rectificación Interna/fisiología
18.
Circ Res ; 94(10): 1332-9, 2004 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-15087421

RESUMEN

The inwardly rectifying potassium (Kir) 2.x channels mediate the cardiac inward rectifier potassium current (I(K1)). In addition to differences in current density, atrial and ventricular I(K1) have differences in outward current profiles and in extracellular potassium ([K+]o) dependence. The whole-cell patch-clamp technique was used to study these properties in heterologously expressed Kir2.x channels and atrial and ventricular I(K1) in guinea pig and sheep hearts. Kir2.x channels showed distinct rectification profiles: Kir2.1 and Kir2.2 rectified completely at potentials more depolarized than -30 mV (I approximately 0 pA). In contrast, rectification was incomplete for Kir2.3 channels. In guinea pig atria, which expressed mainly Kir2.1, I(K1) rectified completely. In sheep atria, which predominantly expressed Kir2.3 channels, I(K1) did not rectify completely. Single-channel analysis of sheep Kir2.3 channels showed a mean unitary conductance of 13.1+/-0.1 pS in 15 cells, which corresponded with I(K1) in sheep atria (9.9+/-0.1 pS in 32 cells). Outward Kir2.1 currents were increased in 10 mmol/L [K+]o, whereas Kir2.3 currents did not increase. Correspondingly, guinea pig (but not sheep) atrial I(K1) showed an increase in outward currents in 10 mmol/L [K+]o. Although the ventricles of both species expressed Kir2.1 and Kir2.3, outward I(K1) currents rectified completely and increased in high [K+]o-displaying Kir2.1-like properties. Likewise, outward current properties of heterologously expressed Kir2.1-Kir2.3 complexes in normal and 10 mmol/L [K+]o were similar to Kir2.1 but not Kir2.3. Thus, unique properties of individual Kir2.x isoforms, as well as heteromeric Kir2.x complexes, determine regional and species differences of I(K1) in the heart.


Asunto(s)
Función Atrial , Canales de Potasio de Rectificación Interna/metabolismo , Función Ventricular , Animales , Línea Celular , Conductividad Eléctrica , Cobayas , Atrios Cardíacos/citología , Ventrículos Cardíacos/citología , Humanos , Miocitos Cardíacos/fisiología , Técnicas de Placa-Clamp , Isoformas de Proteínas/metabolismo , Ovinos , Especificidad de la Especie
19.
Circ Res ; 90(4): 450-7, 2002 Mar 08.
Artículo en Inglés | MEDLINE | ID: mdl-11884375

RESUMEN

Previous studies show that chemical regulation of connexin43 (Cx43) gap junction channels depends on the integrity of the carboxyl terminal (CT) domain. Experiments using Xenopus oocytes show that truncation of the CT domain alters the time course for current inactivation; however, correlation with the behavior of single Cx43 channels has been lacking. Furthermore, whereas chemical gating is associated with a "ball-and-chain" mechanism, there is no evidence whether transjunctional voltage regulation for Cx43 follows a similar model. We provide data on the properties of transjunctional currents from voltage-clamped pairs of mammalian tumor cells expressing either wild-type Cx43 or a mutant of Cx43 lacking the carboxyl terminal domain (Cx43M257). Cx43 transjunctional currents showed bi-exponential decay and a residual steady-state conductance of approximately 35% maximum. Transjunctional currents recorded from Cx43M257 channels displayed a single, slower exponential decay. Long transjunctional voltage pulses caused virtual disappearance of the residual current at steady state. Single channel data revealed disappearance of the residual state, increase in the mean open time, and slowing of the transition times between open and closed states. Coexpression of CxM257 with Cx43CT in a separate fragment restored the lower conductance state. We propose that Cx43CT is an effector of fast voltage gating. Truncation of Cx43CT limits channel transitions to those occurring across the higher energy barrier that separates open and closed states. We further propose that a ball-and-chain interaction provides the fast component of voltage-dependent gating between CT domain and a receptor affiliated with the pore.


Asunto(s)
Conexina 43/metabolismo , Uniones Comunicantes/fisiología , Activación del Canal Iónico/fisiología , Neuroblastoma/metabolismo , Animales , Conexina 43/genética , Electrofisiología , Cinética , Ratones , Mutagénesis Sitio-Dirigida , Oocitos/metabolismo , Técnicas de Placa-Clamp , Tiempo de Reacción , Eliminación de Secuencia , Relación Estructura-Actividad , Transfección , Células Tumorales Cultivadas , Xenopus
20.
Artículo en Inglés | MEDLINE | ID: mdl-27932425

RESUMEN

BACKGROUND: Mutations in SCN2B, encoding voltage-gated sodium channel ß2-subunits, are associated with human cardiac arrhythmias, including atrial fibrillation and Brugada syndrome. Because of this, we propose that ß2-subunits play critical roles in the establishment or maintenance of normal cardiac electric activity in vivo. METHODS AND RESULTS: To understand the pathophysiological roles of ß2 in the heart, we investigated the cardiac phenotype of Scn2b null mice. We observed reduced sodium and potassium current densities in ventricular myocytes, as well as conduction slowing in the right ventricular outflow tract region. Functional reentry, resulting from the interplay between slowed conduction, prolonged repolarization, and increased incidence of premature ventricular complexes, was found to underlie the mechanism of spontaneous polymorphic ventricular tachycardia. Scn5a transcript levels were similar in Scn2b null and wild-type ventricles, as were levels of Nav1.5 protein, suggesting that similar to the previous work in neurons, the major function of ß2-subunits in the ventricle is to chaperone voltage-gated sodium channel α-subunits to the plasma membrane. Interestingly, Scn2b deletion resulted in region-specific effects in the heart. Scn2b null atria had normal levels of sodium current density compared with wild type. Scn2b null hearts were more susceptible to atrial fibrillation, had increased levels of fibrosis, and higher repolarization dispersion than wild-type littermates. CONCLUSIONS: Genetic deletion of Scn2b in mice results in ventricular and atrial arrhythmias, consistent with reported SCN2B mutations in human patients.


Asunto(s)
Fibrilación Atrial/genética , Sistema de Conducción Cardíaco/fisiopatología , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canales de Potasio/genética , Taquicardia Ventricular/genética , Subunidad beta-2 de Canal de Sodio Activado por Voltaje/genética , Potenciales de Acción , Animales , Fibrilación Atrial/fisiopatología , Western Blotting , Células Cultivadas , Eliminación de Gen , Predisposición Genética a la Enfermedad , Ratones , Monocitos , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Taquicardia Ventricular/fisiopatología
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