RESUMO
Background Sodium-calcium (Ca2+) exchanger isoform 1 (NCX1) is the dominant Ca2+ efflux mechanism in cardiomyocytes and is critical to maintaining Ca2+ homeostasis during excitation-contraction coupling. NCX1 activity has been implicated in the pathogenesis of cardiovascular diseases, but a lack of specific NCX1 blockers complicates experimental interpretation. Our aim was to develop a tamoxifen-inducible NCX1 knockout (KO) mouse to investigate compensatory adaptations of acute ablation of NCX1 on excitation-contraction coupling and intracellular Ca2+ regulation, and to examine whether acute KO of NCX1 confers resistance to triggered arrhythmia and ischemia/reperfusion injury. Methods and Results We used the α-myosin heavy chain promoter (Myh6)-MerCreMer promoter to create a tamoxifen-inducible cardiac-specific NCX1 KO mouse. Within 1 week of tamoxifen injection, NCX1 protein expression and current were dramatically reduced. Diastolic Ca2+ increased despite adaptive reductions in Ca2+ current and action potential duration and compensatory increases in excitation-contraction coupling gain, sarcoplasmic reticulum Ca2+ ATPase 2 and plasma membrane Ca2+ ATPase. As these adaptations progressed over 4 weeks, diastolic Ca2+ normalized and SR Ca2+ load increased. Left ventricular function remained normal, but mild fibrosis and hypertrophy developed. Transcriptomics revealed modification of cardiovascular-related gene networks including cell growth and fibrosis. NCX1 KO reduced spontaneous action potentials triggered by delayed afterdepolarizations and reduced scar size in response to ischemia/reperfusion. Conclusions Tamoxifen-inducible NCX1 KO mice adapt to acute genetic ablation of NCX1 by reducing Ca2+ influx, increasing alternative Ca2+ efflux pathways, and increasing excitation-contraction coupling gain to maintain contractility at the cost of mild Ca2+-activated hypertrophy and fibrosis and decreased survival. Nevertheless, KO myocytes are protected against spontaneous action potentials and ischemia/reperfusion injury.
Assuntos
Arritmias Cardíacas , Cálcio , Miócitos Cardíacos , Traumatismo por Reperfusão , Trocador de Sódio e Cálcio , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/prevenção & controle , Cálcio/metabolismo , Fibrose , Hipertrofia , Camundongos , Camundongos Knockout , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Trocador de Sódio e Cálcio/genética , Tamoxifeno/farmacologiaRESUMO
Na/Ca exchange is the dominant calcium (Ca) efflux mechanism in cardiac myocytes. Although our knowledge of exchanger function (NCX1 in the heart) was originally established using biochemical and electrophysiological tools such as cardiac sarcolemmal vesicles and the giant patch technique [1-4], many advances in our understanding of the physiological/pathophysiological roles of NCX1 in the heart have been obtained using a suite of genetically modified mice. Early mouse studies focused on modification of expression levels of NCX1 in the ventricles, with transgenic overexpressors, global NCX1 knockout (KO) mice (which were embryonic lethal if homozygous), and finally ventricular-specific NCX1 KO [5-12]. We found, to our surprise, that ventricular cardiomyocytes lacking NCX1 can survive and function by engaging a clever set of adaptations to minimize Ca entry, while maintaining contractile function through an increase in excitation-contraction (EC) coupling gain [5,6,13]. Having studied ventricular NCX1 ablation in detail, we more recently focused on elucidating the role of NCX1 in the atria through altering NCX1 expression. Using a novel atrial-specific NCX1 KO mouse, we found unexpected changes in atrial cell morphology and calcium handling, together with dramatic alterations in the function of sinoatrial node (SAN) pacemaker activity. In this review, we will discuss these findings and their implications for cardiac disease.
Assuntos
Relógios Biológicos , Cálcio/metabolismo , Acoplamento Excitação-Contração , Átrios do Coração/metabolismo , Nó Sinoatrial/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Sódio/metabolismo , Animais , HumanosRESUMO
Transverse-axial tubules (TATs) are commonly assumed to be sparse or absent in atrial myocytes from small animals. Atrial myocytes from rats, cats and rabbits lack TATs, which results in a characteristic "V"-shaped Ca release pattern in confocal line-scan recordings due to the delayed rise of Ca in the center of the cell. To examine TAT expression in isolated mouse atrial myocytes, we loaded them with the membrane dye Di-4-ANEPPS to label TATs. We found that >80% of atrial myocytes had identifiable TATs. Atria from male mice had a higher TAT density than female mice, and TAT density correlated with cell width. Using the fluorescent Ca indicator Fluo-4-AM and confocal imaging, we found that wild type (WT) mouse atrial myocytes generate near-synchronous Ca transients, in contrast to the "V"-shaped pattern typically reported in other small animals such as rat. In atrial-specific Na-Ca exchanger (NCX) knockout (KO) mice, which develop sinus node dysfunction and atrial hypertrophy with dilation, we found a substantial loss of atrial TATs in isolated atrial myocytes. There was a greater loss of transverse tubules compared to axial tubules, resulting in a dominance of axial tubules. Consistent with the overall loss of TATs, NCX KO atrial myocytes displayed a "V"-shaped Ca transient with slower and reduced central (CT) Ca release and uptake in comparison to subsarcolemmal (SS) Ca release. We compared chemically detubulated (DT) WT cells to KO, and found similar slowing of CT Ca release and uptake. However, SS Ca transients in the WT DT cells had faster uptake kinetics than KO cells, consistent with the presence of NCX and normal sarcolemmal Ca efflux in the WT DT cells. We conclude that the remodeling of NCX KO atrial myocytes is accompanied by a loss of TATs leading to abnormal Ca release and uptake that could impact atrial contractility and rhythm.
Assuntos
Átrios do Coração/metabolismo , Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/genética , Animais , Remodelamento Atrial/genética , Cálcio/metabolismo , Sinalização do Cálcio , Modelos Animais de Doenças , Acoplamento Excitação-Contração , Feminino , Átrios do Coração/patologia , Átrios do Coração/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Imagem Molecular , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Trocador de Sódio e Cálcio/metabolismoRESUMO
KEY POINTS: Repolarizing currents through K+ channels are essential for proper sinoatrial node (SAN) pacemaking, but the influence of intracellular Ca2+ on repolarization in the SAN is uncertain. We identified all three isoforms of Ca2+ -activated small conductance K+ (SK) channels in the murine SAN. SK channel blockade slows repolarization and subsequent depolarization of SAN cells. In the atrial-specific Na+ /Ca2+ exchanger (NCX) knockout mouse, cellular Ca2+ accumulation during spontaneous SAN pacemaker activity produces intermittent hyperactivation of SK channels, leading to arrhythmic pauses alternating with bursts of pacing. These findings suggest that Ca2+ -sensitive SK channels can translate changes in cellular Ca2+ into a repolarizing current capable of modulating pacemaking. SK channels are a potential pharmacological target for modulating SAN rate or treating SAN dysfunction, particularly under conditions characterized by abnormal increases in diastolic Ca2+ . ABSTRACT: Small conductance K+ (SK) channels have been implicated as modulators of spontaneous depolarization and electrical conduction that may be involved in cardiac arrhythmia. However, neither their presence nor their contribution to sinoatrial node (SAN) pacemaker activity has been investigated. Using quantitative PCR (q-PCR), immunostaining and patch clamp recordings of membrane current and voltage, we identified all three SK isoforms (SK1, SK2 and SK3) in mouse SAN. Inhibition of SK channels with the specific blocker apamin prolonged action potentials (APs) in isolated SAN cells. Apamin also slowed diastolic depolarization and reduced pacemaker rate in isolated SAN cells and intact tissue. We investigated whether the Ca2+ -sensitive nature of SK channels could explain arrhythmic SAN pacemaker activity in the atrial-specific Na+ /Ca2+ exchange (NCX) knockout (KO) mouse, a model of cellular Ca2+ overload. SAN cells isolated from the NCX KO exhibited higher SK current than wildtype (WT) and apamin prolonged their APs. SK blockade partially suppressed the arrhythmic burst pacing pattern of intact NCX KO SAN tissue. We conclude that SK channels have demonstrable effects on SAN pacemaking in the mouse. Their Ca2+ -dependent activation translates changes in cellular Ca2+ into a repolarizing current capable of modulating regular pacemaking. This Ca2+ dependence also promotes abnormal automaticity when these channels are hyperactivated by elevated Ca2+ . We propose SK channels as a potential target for modulating SAN rate, and for treating patients affected by SAN dysfunction, particularly in the setting of Ca2+ overload.
Assuntos
Relógios Biológicos/fisiologia , Cálcio/metabolismo , Nó Sinoatrial/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Apamina/farmacologia , Relógios Biológicos/efeitos dos fármacos , Feminino , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/metabolismo , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Masculino , Camundongos , Camundongos Knockout , Isoformas de Proteínas/metabolismo , Nó Sinoatrial/efeitos dos fármacosRESUMO
Three different Na+/Ca2+ exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, are expressed in brain where they play a relevant role in maintaining Na+ and Ca2+ homeostasis. Although the neuroprotective roles of NCX2 and NCX3 in stroke have been elucidated, the relevance of NCX1 is still unknown because of embryonic lethality of its knocking-out, heart dysfunctions when it is overexpressed, and the lack of selectivity in currently available drugs. To overcome these limitations we generated two conditional genetically modified mice that upon tamoxifen administration showed a selective decrease or increase of NCX1 in cortical and hippocampal neurons. Interestingly, in cortex and hippocampus NCX1 overexpression increased, where NCX1 knock-out reduced, both exchanger activity and Akt1 phosphorylation, a neuronal survival signaling. More important, mice overexpressing NCX1 showed a reduced ischemic volume and an amelioration of focal and general deficits when subjected to transient middle cerebral artery occlusion. Conversely, NCX1-knock-out mice displayed a worsening of brain damage, focal and neurological deficits with a decrease in Akt phosphorylation. These results support the idea that NCX1 overexpression/activation may represent a feasible therapeutic opportunity in stroke intervention.
Assuntos
Córtex Cerebral/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Acidente Vascular Cerebral/metabolismo , Animais , Córtex Cerebral/patologia , Modelos Animais de Doenças , Regulação para Baixo , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Hipocampo/patologia , Masculino , Camundongos Knockout , Neurônios/patologia , Fosforilação , Trocador de Sódio e Cálcio/genética , Acidente Vascular Cerebral/genética , Tamoxifeno/administração & dosagem , Tamoxifeno/farmacologia , Regulação para CimaRESUMO
In sinoatrial node (SAN) cells, electrogenic sodium-calcium exchange (NCX) is the dominant calcium (Ca) efflux mechanism. However, the role of NCX in the generation of SAN automaticity is controversial. To investigate the contribution of NCX to pacemaking in the SAN, we performed optical voltage mapping and high-speed 2D laser scanning confocal microscopy (LSCM) of Ca dynamics in an ex vivo intact SAN/atrial tissue preparation from atrial-specific NCX knockout (KO) mice. These mice lack P waves on electrocardiograms, and isolated NCX KO SAN cells are quiescent. Voltage mapping revealed disorganized and arrhythmic depolarizations within the NCX KO SAN that failed to propagate into the atria. LSCM revealed intermittent bursts of Ca transients. Bursts were accompanied by rising diastolic Ca, culminating in long pauses dominated by Ca waves. The L-type Ca channel agonist BayK8644 reduced the rate of Ca transients and inhibited burst generation in the NCX KO SAN whereas the Ca buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl ester) (BAPTA AM) did the opposite. These results suggest that cellular Ca accumulation hinders spontaneous depolarization in the NCX KO SAN, possibly by inhibiting L-type Ca currents. The funny current (If) blocker ivabradine also suppressed NCX KO SAN automaticity. We conclude that pacemaker activity is present in the NCX KO SAN, generated by a mechanism that depends upon If. However, the absence of NCX-mediated depolarization in combination with impaired Ca efflux results in intermittent bursts of pacemaker activity, reminiscent of human sinus node dysfunction and "tachy-brady" syndrome.
Assuntos
Potenciais de Ação , Relógios Biológicos , Nó Sinoatrial/fisiologia , Trocador de Sódio e Cálcio/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Conexinas/metabolismo , Diástole , Estimulação Elétrica , Feminino , Fibrose , Espaço Intracelular/metabolismo , Masculino , Camundongos Knockout , Receptores Adrenérgicos beta/metabolismoRESUMO
KEY POINTS: Inositol-1,4,5-trisphosphate receptors (IP3 Rs) modulate pacemaking in embryonic heart, but their role in adult sinoatrial node (SAN) pacemaking is uncertain. We found that stimulation of IP3 Rs accelerates spontaneous pacing rate in isolated mouse SAN cells, whereas inhibition of IP3 Rs slows pacing. In atrial-specific sodium-calcium exchanger (NCX) knockout (KO) SAN cells, where the Ca(2+) clock is uncoupled from the membrane clock, IP3 R agonists and antagonists modulate the rate of spontaneous Ca(2+) waves, suggesting that IP3 R-mediated Ca(2+) release modulates the Ca(2+) clock. IP3 R modulation also regulates Ca(2+) spark parameters, a reflection of ryanodine receptor open probability, consistent with the effect of IP3 signalling on Ca(2+) clock frequency. Modulation of Ca(2+) clock frequency by IP3 signalling in NCX KO SAN cells demonstrates that the effect is independent of NCX. These findings support development of IP3 signalling modulators for regulation of heart rate, particularly in heart failure where IP3 Rs are upregulated. ABSTRACT: Cardiac pacemaking initiated by the sinus node is attributable to the interplay of several membrane currents. These include the depolarizing 'funny current' (If ) and the sodium-calcium exchanger current (INCX ). The latter is activated by ryanodine receptor (RyR)-mediated calcium (Ca(2+) ) release from the sarcoplasmic reticulum (SR). Another SR Ca(2+) release channel, the inositol-1,4,5-triphosphate receptor (IP3 R), has been implicated in the generation of spontaneous Ca(2+) release in atrial and ventricular cardiomyocytes. Whether IP3 R-mediated Ca(2+) release also influences SAN automaticity is controversial, in part due to the confounding influence of periodic Ca(2+) flux through the sarcolemma accompanying each beat. We took advantage of atrial-specific sodium-calcium exchanger (NCX) knockout (KO) SAN cells to study the influence of IP3 signalling on cardiac pacemaking in a system where periodic intracellular Ca(2+) cycling persists despite the absence of depolarization or Ca(2+) flux across the sarcolemma. We recorded confocal line scans of spontaneous Ca(2+) release in WT and NCX KO SAN cells in the presence or absence of an IP3 R blocker (2-aminoethoxydiphenyl borate, 2-APB), or during block of IP3 production by the phospholipase C inhibitor U73122. 2-APB and U73122 decreased the frequency of spontaneous Ca(2+) transients and waves in WT and NCX KO cells, respectively. Alternatively, increased IP3 production induced by phenylephrine increased Ca(2+) transient and wave frequency. We conclude that IP3 R-mediated SR Ca(2+) flux is crucial for initiating and modulating the RyR-mediated Ca(2+) cycling that regulates SAN pacemaking. Our results in NCX KO SAN cells also demonstrate that RyRs, but not NCX, are required for IP3 to modulate Ca(2+) clock frequency.
Assuntos
Relógios Biológicos/fisiologia , Cálcio/fisiologia , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Nó Sinoatrial/citologia , Animais , Feminino , Receptores de Inositol 1,4,5-Trifosfato/agonistas , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Masculino , Camundongos Knockout , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/fisiologiaRESUMO
Unregulated Ca(2+) entry is thought to underlie muscular dystrophy. Here, we generated skeletal-muscle-specific transgenic (TG) mice expressing the Na(+)-Ca(2+) exchanger 1 (NCX1) to model its identified augmentation during muscular dystrophy. The NCX1 transgene induced dystrophy-like disease in all hind-limb musculature, as well as exacerbated the muscle disease phenotypes in δ-sarcoglycan (Sgcd(-/-)), Dysf(-/-), and mdx mouse models of muscular dystrophy. Antithetically, muscle-specific deletion of the Slc8a1 (NCX1) gene diminished hind-limb pathology in Sgcd(-/-) mice. Measured increases in baseline Na(+) and Ca(2+) in dystrophic muscle fibers of the hind-limb musculature predicts a net Ca(2+) influx state due to reverse-mode operation of NCX1, which mediates disease. However, the opposite effect is observed in the diaphragm, where NCX1 overexpression mildly protects from dystrophic disease through a predicted enhancement in forward-mode NCX1 operation that reduces Ca(2+) levels. Indeed, Atp1a2(+/-) (encoding Na(+)-K(+) ATPase α2) mice, which have reduced Na(+) clearance rates that would favor NCX1 reverse-mode operation, showed exacerbated disease in the hind limbs of NCX1 TG mice, similar to treatment with the Na(+)-K(+) ATPase inhibitor digoxin. Treatment of Sgcd(-/-) mice with ranolazine, a broadly acting Na(+) channel inhibitor that should increase NCX1 forward-mode operation, reduced muscular pathology.
Assuntos
Cálcio/metabolismo , Distrofia Muscular Animal/genética , Trocador de Sódio e Cálcio/metabolismo , Sódio/metabolismo , Acetanilidas/farmacologia , Animais , Digoxina/farmacologia , Disferlina , Inibidores Enzimáticos/farmacologia , Membro Posterior/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Knockout , Músculo Esquelético/patologia , Piperazinas/farmacologia , Ranolazina , Sarcoglicanas/genética , Bloqueadores dos Canais de Sódio/farmacologia , Trocador de Sódio e Cálcio/genética , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
The origin of sinoatrial node (SAN) pacemaker activity in the heart is controversial. The leading candidates are diastolic depolarization by "funny" current (If) through HCN4 channels (the "Membrane Clock" hypothesis), depolarization by cardiac Na-Ca exchange (NCX1) in response to intracellular Ca cycling (the "Calcium Clock" hypothesis), and a combination of the two ("Coupled Clock"). To address this controversy, we used Cre/loxP technology to generate atrial-specific NCX1 KO mice. NCX1 protein was undetectable in KO atrial tissue, including the SAN. Surface ECG and intracardiac electrograms showed no atrial depolarization and a slow junctional escape rhythm in KO that responded appropriately to ß-adrenergic and muscarinic stimulation. Although KO atria were quiescent they could be stimulated by external pacing suggesting that electrical coupling between cells remained intact. Despite normal electrophysiological properties of If in isolated patch clamped KO SAN cells, pacemaker activity was absent. Recurring Ca sparks were present in all KO SAN cells, suggesting that Ca cycling persists but is uncoupled from the sarcolemma. We conclude that NCX1 is required for normal pacemaker activity in murine SAN.
Assuntos
Cálcio/metabolismo , Átrios do Coração/metabolismo , Nó Sinoatrial/fisiologia , Sódio/metabolismo , Animais , Transporte de Íons , Camundongos , Camundongos Knockout , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/fisiologiaRESUMO
Sodium-calcium exchange (NCX) is the major calcium (Ca) efflux mechanism of ventricular cardiomyocytes. Consequently the exchanger plays a critical role in the regulation of cellular Ca content and hence contractility. Reductions in Ca efflux by the exchanger, such as those produced by elevated intracellular sodium (Na) in response to cardiac glycosides, raise sarcoplasmic reticulum (SR) Ca stores. The result is an increased Ca transient and cardiac contractility. Enhanced Ca efflux activity by the exchanger, for example during heart failure, may reduce diadic cleft Ca and excitation-contraction (EC) coupling gain. This aggravates the impaired contractility associated with SR Ca ATPase dysfunction and reduced SR Ca load in failing heart muscle. Recent data from our laboratories indicate that NCX can also impact the efficiency of EC coupling and contractility independent of SR Ca load through diadic cleft priming with Ca during the upstroke of the action potential. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".
Assuntos
Cálcio/metabolismo , Acoplamento Excitação-Contração , Contração Miocárdica , Sódio/metabolismo , Potenciais de Ação , Animais , Transporte Biológico , Estruturas da Membrana Celular/metabolismo , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Humanos , Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismoRESUMO
The topology of the plasma membrane Na(+)/Ca(2+) exchanger of cardiac muscle, NCX1, is uncertain. Biochemical analyses have indicated the presence of 9 transmembrane segments (TMSs) whereas the recent crystal structure of a prokaryotic homologue has 10 TMSs. The discrepancy is towards the C-terminus of the proteins where the prokaryotic homologue has an additional TMS8. To resolve this apparent disagreement, we re-assessed the topology of the C-terminal TMSs of NCX1. We examined the ability of internal or external cysteine residues in the N-terminal portion of NCX1 to crosslink with cysteine residues, of uncertain orientation, in the C-terminal portion of the protein. The results strongly support a model of NCX1 with 10 TMSs as found in the prokaryotic homologue.
Assuntos
Trocador de Sódio e Cálcio/química , Animais , Linhagem Celular , Reagentes de Ligações Cruzadas/química , Etilenoglicóis/química , Humanos , Modelos Moleculares , Mariposas , Fenantrolinas/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ácidos Tiossulfônicos/químicaRESUMO
The Na(+)/Ca(2+) exchanger protein was first isolated from cardiac sarcolemma in 1988 and cloned in 1990. This allowed study of Na(+)/Ca(2+) exchange at the molecular level to begin. I will review the story leading to the cloning of NCX and the research that resulted from this event. This will include structure-function studies such as determination of the numbers of transmembrane segments and topological arrangement. Information on ion transport sites has been gathered from site-directed mutagenesis. The regions involved in Ca(2+) regulation have been identified, analyzed, and crystallized.We have also generated genetically altered mice to study the role of NCX in the myocardium. Of special interest are mice with atrial- or ventricular-specific KO of NCX that reveal new information on the role of NCX in excitation-contraction coupling and in cardiac pacemaker activity.
Assuntos
Relógios Biológicos/fisiologia , Clonagem Molecular , Proteínas Musculares , Miocárdio , Sarcolema , Trocador de Sódio e Cálcio , Animais , Aniversários e Eventos Especiais , Pesquisa Biomédica/história , História do Século XX , História do Século XXI , Humanos , Transporte de Íons , Camundongos , Camundongos Transgênicos , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas Musculares/isolamento & purificação , Proteínas Musculares/metabolismo , Mutagênese Sítio-Dirigida , Miocárdio/química , Miocárdio/metabolismo , Estrutura Secundária de Proteína , Sarcolema/química , Sarcolema/metabolismo , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/isolamento & purificação , Trocador de Sódio e Cálcio/metabolismoRESUMO
The plasma membrane Na(+)/Ca(2+) exchanger (NCX) plays a critical role in the maintenance of Ca(2+) homeostasis in a variety of tissues. NCX accomplishes this task by either lowering or increasing the intracellular Ca(2+) concentration, a process which depends on electrochemical gradients. During each cycle, three Na(+) are transported in the opposite direction to one Ca(2+), resulting in an electrogenic transport that can be measured as an ionic current.The residues involved in ion translocation are unknown. A residue thought to be important for Na(+) and/or Ca(2+) transport, Ser(110), was replaced with a cysteine, and the properties of the resulting exchanger mutant were analyzed using the giant patch technique. Data indicate that this residue, located in transmembrane segment 2 (part of the α-1 repeat), is important for both Na(+) and Ca(2+) translocations. Using cysteine susceptibility analysis, we demonstrated that Ser(110) is exposed to the cytoplasm when the exchanger is in the inward state configuration.
Assuntos
Cálcio , Homeostase/fisiologia , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/metabolismo , Sódio , Animais , Cálcio/química , Cálcio/metabolismo , Humanos , Transporte de Íons/fisiologia , Mutação , Estrutura Secundária de Proteína , Sódio/química , Sódio/metabolismo , Trocador de Sódio e Cálcio/genéticaRESUMO
Cardiovascular disease is a leading cause of death worldwide, with ischemic heart disease alone accounting for >12% of all deaths, more than HIV/AIDS, tuberculosis, lung, and breast cancer combined. Heart disease has been the leading cause of death in the United States for the past 85 years and is a major cause of disability and health-care expenditures. The cardiac conditions most likely to result in death include heart failure and arrhythmias, both a consequence of ischemic coronary disease and myocardial infarction, though chronic hypertension and valvular diseases are also important causes of heart failure. Sodium-calcium exchange (NCX) is the dominant calcium (Ca2+) efflux mechanism in cardiac cells. Using ventricular-specific NCX knockout mice, we have found that NCX is also an essential regulator of cardiac contractility independent of sarcoplasmic reticulum Ca2+ load. During the upstroke of the action potential, sodium (Na+) ions enter the diadic cleft space between the sarcolemma and the sarcoplasmic reticulum. The rise in cleft Na+, in conjunction with depolarization, causes NCX to transiently reverse. Ca2+ entry by this mechanism then "primes" the diadic cleft so that subsequent Ca2+ entry through Ca2+ channels can more efficiently trigger Ca2+ release from the sarcoplasmic reticulum. In NCX knockout mice, this mechanism is inoperative (Na+ current has no effect on the Ca2+ transient), and excitation-contraction coupling relies upon the elevated diadic cleft Ca2+ that arises from the slow extrusion of cytoplasmic Ca2+ by the ATP-dependent sarcolemmal Ca2+ pump. Thus, our data support the conclusion that NCX is an important regulator of cardiac contractility. These findings suggest that manipulation of NCX may be beneficial in the treatment of heart failure.
Assuntos
Cálcio/metabolismo , Insuficiência Cardíaca/metabolismo , Proteínas Musculares/metabolismo , Contração Miocárdica , Miocárdio/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Feminino , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/terapia , Humanos , Masculino , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Miocárdio/patologia , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/patologia , Trocador de Sódio e Cálcio/genéticaRESUMO
Previous studies have shown that digitalis drugs, acting as specific inhibitors of cardiac Na(+)/K(+)-ATPase, not only cause positive inotropic effects, but also activate cell signaling pathways that lead to cardiac myocyte hypertrophy. A major aim of this work was to assess the role of Na(+)/Ca(2+)-exchanger, NCX1, in the above two seemingly related drug effects. Using a mouse with ventricular-specific knockout (KO) of NCX1, ouabain-induced positive inotropy that was evident in isolated wild-type (Wt) hearts was clearly reduced in KO hearts. Ouabain also increased Ca(2+) transient amplitudes in Wt myocytes, but not in KO myocytes. Ouabain-induced activations of ERK 1/2 were noted in Wt myocytes, but not in KO myocytes; however, ouabain activated PI3K1A and Akt in both Wt and KO myocytes. Protein synthesis rate, as a measure of hypertrophy, was increased by ouabain in Wt and KO myocytes; these drug effects were prevented by a PI3K inhibitor but not by a MEK/ERK inhibitor. Hypertrophy caused by ET-1, but not that induced by ouabain, was accompanied by upregulation of BNP gene in Wt and KO myocytes. The findings indicate 1) the necessity of NCX1 for positive inotropic action of ouabain; 2) the irrelevance of NCX1 and ERK 1/2 activation to ouabain-induced hypertrophy; and 3) that hypertrophy caused by ouabain through the activation of PI3K1A/Akt pathway is likely to be beneficial to the heart.
Assuntos
Cardiomegalia/fisiopatologia , Cardiotônicos/farmacologia , Inibidores Enzimáticos/farmacologia , Ouabaína/farmacologia , Transdução de Sinais/efeitos dos fármacos , Trocador de Sódio e Cálcio/fisiologia , Animais , Cálcio/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas In Vitro , Masculino , Camundongos , Contração Miocárdica/efeitos dos fármacos , Miocárdio/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Trocador de Sódio e Cálcio/genética , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
RATIONALE: The sodium-calcium exchanger 1 (NCX1) is predominantly expressed in the heart and is implicated in controlling automaticity in isolated sinoatrial node (SAN) pacemaker cells, but the potential role of NCX1 in determining heart rate in vivo is unknown. OBJECTIVE: To determine the role of Ncx1 in heart rate. METHODS AND RESULTS: We used global myocardial and SAN-targeted conditional Ncx1 knockout (Ncx1(-/-)) mice to measure the effect of the NCX current on pacemaking activity in vivo, ex vivo, and in isolated SAN cells. We induced conditional Ncx1(-/-) using a Cre/loxP system. Unexpectedly, in vivo and ex vivo hearts and isolated SAN cells showed that basal rates in Ncx1(-/-) (retaining ≈20% of control level NCX current) and control mice were similar, suggesting that physiological NCX1 expression is not required for determining resting heart rate. However, increases in heart rate and SAN cell automaticity in response to isoproterenol or the dihydropyridine Ca(2+) channel agonist BayK8644 were significantly blunted or eliminated in Ncx1(-/-) mice, indicating that NCX1 is important for fight or flight heart rate responses. In contrast, the pacemaker current and L-type Ca(2+) currents were equivalent in control and Ncx1(-/-) SAN cells under resting and isoproterenol-stimulated conditions. Ivabradine, a pacemaker current antagonist with clinical efficacy, reduced basal SAN cell automaticity similarly in control and Ncx1(-/-) mice. However, ivabradine decreased automaticity in SAN cells isolated from Ncx1(-/-) mice more effectively than in control SAN cells after isoproterenol, suggesting that the importance of NCX current in fight or flight rate increases is enhanced after pacemaker current inhibition. CONCLUSIONS: Physiological Ncx1 expression is required for increasing sinus rates in vivo, ex vivo, and in isolated SAN cells, but not for maintaining resting heart rate.
Assuntos
Frequência Cardíaca/fisiologia , Descanso/fisiologia , Nó Sinoatrial/fisiologia , Trocador de Sódio e Cálcio/antagonistas & inibidores , Trocador de Sódio e Cálcio/genética , Agonistas Adrenérgicos beta/farmacologia , Animais , Frequência Cardíaca/efeitos dos fármacos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Nó Sinoatrial/citologia , Nó Sinoatrial/efeitos dos fármacos , Trocador de Sódio e Cálcio/metabolismo , Trocador de Sódio e Cálcio/fisiologiaRESUMO
The cardiac Na(+)/Ca(2+) exchanger (NCX) generates an inward electrical current during SR-Ca(2+) release, thus possibly promoting afterdepolarizations of the action potential (AP). We used transgenic mice 12.5 weeks or younger with cardiomyocyte-directed overexpression of NCX (NCX-Tg) to study the proarrhythmic potential and mechanisms of enhanced NCX activity. NCX-Tg exhibited normal echocardiographic left ventricular function and heart/body weight ratio, while the QT interval was prolonged in surface ECG recordings. Langendorff-perfused NCX-Tg, but not wild-type (WT) hearts, developed ventricular tachycardia. APs and ionic currents were measured in isolated cardiomyocytes. Cell capacitance was unaltered between groups. APs were prolonged in NCX-Tg versus WT myocytes along with voltage-activated K(+) currents (K(v)) not being reduced but even increased in amplitude. During abrupt changes in pacing cycle length, early afterdepolarizations (EADs) were frequently recorded in NCX-Tg but not in WT myocytes. Next to EADs, delayed afterdepolarizations (DAD) triggering spontaneous APs (sAPs) occurred in NCX-Tg but not in WT myocytes. To test whether sAPs were associated with spontaneous Ca(2+) release (sCR), Ca(2+) transients were recorded. Despite the absence of sAPs in WT, sCR was observed in myocytes of both genotypes suggesting a facilitated translation of sCR into DADs in NCX-Tg. Moreover, sCR was more frequent in NCX-Tg as compared to WT. Myocardial protein levels of Ca(2+)-handling proteins were not different between groups except the ryanodine receptor (RyR), which was increased in NCX-Tg versus WT. We conclude that NCX overexpression is proarrhythmic in a non-failing environment even in the absence of reduced K(V). The underlying mechanisms are: (1) occurrence of EADs due to delayed repolarization; (2) facilitated translation from sCR into DADs; (3) proneness to sCR possibly caused by altered Ca(2+) handling and/or increased RyR expression.
Assuntos
Potenciais de Ação/fisiologia , Arritmias Cardíacas/metabolismo , Coração/fisiologia , Proteínas de Homeodomínio/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Arritmias Cardíacas/genética , Western Blotting , Modelos Animais de Doenças , Eletrocardiografia , Proteínas de Homeodomínio/genética , Camundongos , Técnicas de Cultura de ÓrgãosRESUMO
The superfamily of cation/Ca(2+) exchangers includes both Na(+)/Ca(2+) exchangers (NCXs) and Na(+)/Ca(2+),K(+) exchangers (NCKX) as the families characterized in most detail. These Ca(2+) transporters have prominent physiological roles. For example, NCX and NCKX are important in regulation of cardiac contractility and visual processes, respectively. The superfamily also has a large number of members of the YrbG family expressed in prokaryotes. However, no members of this family have been functionally expressed, and their transport properties are unknown. We have expressed, purified, and characterized a member of the YrbG family, MaX1 from Methanosarcina acetivorans. MaX1 catalyzes Ca(2+) uptake into membrane vesicles. The Ca(2+) uptake requires intravesicular Na(+) and is stimulated by an inside positive membrane potential. Despite very limited sequence similarity, MaX1 is a Na(+)/Ca(2+) exchanger with kinetic properties similar to those of NCX. The availability of a prokaryotic Na(+)/Ca(2+) exchanger should facilitate structural and mechanistic investigations.
Assuntos
Proteínas Arqueais/química , Methanosarcina/química , Trocador de Sódio e Cálcio/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Cálcio/química , Cálcio/metabolismo , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Expressão Gênica , Transporte de Íons/fisiologia , Potenciais da Membrana/fisiologia , Methanosarcina/genética , Methanosarcina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sódio/química , Sódio/metabolismo , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismo , Relação Estrutura-AtividadeRESUMO
Cytoplasmic Ca(2+) is known to regulate Na(+)-Ca(2+) exchanger (NCX) activity by binding to two adjacent Ca(2+)-binding domains (CBD1 and CBD2) located in the large intracellular loop between transmembrane segments 5 and 6. We investigated Ca(2+)-dependent movements as changes in FRET between exchanger proteins tagged with CFP or YFP at position 266 within the large cytoplasmic loop. Data indicate that the exchanger assembles as a dimer in the plasma membrane. Addition of Ca(2+) decreases the distance between the cytoplasmic loops of NCX pairs. The Ca(2+)-dependent movements detected between paired NCXs were abolished by mutating the Ca(2+) coordination sites in CBD1 (D421A, E451A, and D500V), whereas disruption of the primary Ca(2+) coordination site in CBD2 (E516L) had no effect. Thus, the Ca(2+)-induced conformational changes of NCX dimers arise from the movement of CBD1. FRET studies of CBD1, CBD2, and CBD1-CBD2 peptides displayed Ca(2+)-dependent movements with different apparent affinities. CBD1-CBD2 showed a Ca(2+)-dependent phenotype mirroring full-length NCX but distinct from both CBD1 and CBD2.