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1.
Elife ; 112022 12 14.
Artículo en Inglés | MEDLINE | ID: mdl-36515265

RESUMEN

Adult (3 month) mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TGAC8) adapt to an increased cAMP-induced cardiac workload (~30% increases in heart rate, ejection fraction and cardiac output) for up to a year without signs of heart failure or excessive mortality. Here, we show classical cardiac hypertrophy markers were absent in TGAC8, and that total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TGAC8 was encased by thicker LV walls harboring an increased number of small cardiac myocytes, and a network of small interstitial proliferative non-cardiac myocytes compared to wild type (WT) littermates; Protein synthesis, proteosome activity, and autophagy were enhanced in TGAC8 vs WT, and Nrf-2, Hsp90α, and ACC2 protein levels were increased. Despite increased energy demands in vivo LV ATP and phosphocreatine levels in TGAC8 did not differ from WT. Unbiased omics analyses identified more than 2,000 transcripts and proteins, comprising a broad array of biological processes across multiple cellular compartments, which differed by genotype; compared to WT, in TGAC8 there was a shift from fatty acid oxidation to aerobic glycolysis in the context of increased utilization of the pentose phosphate shunt and nucleotide synthesis. Thus, marked overexpression of AC8 engages complex, coordinate adaptation "circuity" that has evolved in mammalian cells to defend against stress that threatens health or life (elements of which have already been shown to be central to cardiac ischemic pre-conditioning and exercise endurance cardiac conditioning) that may be of biological significance to allow for proper healing in disease states such as infarction or failure of the heart.


Asunto(s)
Adaptación Fisiológica , Miocitos Cardíacos , Estrés Fisiológico , Animales , Ratones , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/fisiopatología , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Hipertrofia/fisiopatología , Ratones Transgénicos , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Humanos
2.
Cells ; 10(11)2021 11 10.
Artículo en Inglés | MEDLINE | ID: mdl-34831329

RESUMEN

Spontaneous AP (action potential) firing of sinoatrial nodal cells (SANC) is critically dependent on protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent protein phosphorylation, which are required for the generation of spontaneous, diastolic local Ca2+ releases (LCRs). Although phosphoprotein phosphatases (PP) regulate protein phosphorylation, the expression level of PPs and phosphatase inhibitors in SANC and the impact of phosphatase inhibition on the spontaneous LCRs and other players of the oscillatory coupled-clock system is unknown. Here, we show that rabbit SANC express both PP1, PP2A, and endogenous PP inhibitors I-1 (PPI-1), dopamine and cyclic adenosine 3',5'-monophosphate (cAMP)-regulated phosphoprotein (DARPP-32), kinase C-enhanced PP1 inhibitor (KEPI). Application of Calyculin A, (CyA), a PPs inhibitor, to intact, freshly isolated single SANC: (1) significantly increased phospholamban (PLB) phosphorylation (by 2-3-fold) at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites, in a time and concentration dependent manner; (2) increased ryanodine receptor (RyR) phosphorylation at the Ser2809 site; (3) substantially increased sarcoplasmic reticulum (SR) Ca2+ load; (4) augmented L-type Ca2+ current amplitude; (5) augmented LCR's characteristics and decreased LCR period in intact and permeabilized SANC, and (6) increased the spontaneous basal AP firing rate. In contrast, the selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on spontaneous AP firing, LCR parameters, or PLB phosphorylation. Application of purified PP1 to permeabilized SANC suppressed LCR, whereas purified PP2A had no effect on LCR characteristics. Our numerical model simulations demonstrated that PP inhibition increases AP firing rate via a coupled-clock mechanism, including respective increases in the SR Ca2+ pumping rate, L-type Ca2+ current, and Na+/Ca2+-exchanger current. Thus, PP1 and its endogenous inhibitors modulate the basal spontaneous firing rate of cardiac pacemaker cells by suppressing SR Ca2+ cycling protein phosphorylation, the SR Ca2+ load and LCRs, and L-type Ca2+ current.


Asunto(s)
Relojes Biológicos , Fosfoproteínas Fosfatasas/metabolismo , Nodo Sinoatrial/citología , Potenciales de Acción/efectos de los fármacos , Animales , Relojes Biológicos/efectos de los fármacos , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Proteínas de Unión al Calcio/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Simulación por Computador , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ventrículos Cardíacos/citología , Toxinas Marinas/farmacología , Modelos Biológicos , Oxazoles/farmacología , Fosforilación/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos
3.
Front Physiol ; 12: 612770, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34566668

RESUMEN

Ca2+ and V m transitions occurring throughout action potential (AP) cycles in sinoatrial nodal (SAN) cells are cues that (1) not only regulate activation states of molecules operating within criticality (Ca2+ domain) and limit-cycle (V m domain) mechanisms of a coupled-clock system that underlies SAN cell automaticity, (2) but are also regulated by the activation states of the clock molecules they regulate. In other terms, these cues are both causes and effects of clock molecular activation (recursion). Recently, we demonstrated that Ca2+ and V m transitions during AP cycles in single SAN cells isolated from mice, guinea pigs, rabbits, and humans are self-similar (obey a power law) and are also self-similar to trans-species AP firing intervals (APFIs) of these cells in vitro, to heart rate in vivo, and to body mass. Neurotransmitter stimulation of ß-adrenergic receptor or cholinergic receptor-initiated signaling in SAN cells modulates their AP firing rate and rhythm by impacting on the degree to which SAN clocks couple to each other, creating the broad physiologic range of SAN cell mean APFIs and firing interval variabilities. Here we show that Ca2+ and V m domain kinetic transitions (time to AP ignition in diastole and 90% AP recovery) occurring within given AP, the mean APFIs, and APFI variabilities within the time series of APs in 230 individual SAN cells are self-similar (obey power laws). In other terms, these long-range correlations inform on self-similar distributions of order among SAN cells across the entire broad physiologic range of SAN APFIs, regardless of whether autonomic receptors of these cells are stimulated or not and regardless of the type (adrenergic or cholinergic) of autonomic receptor stimulation. These long-range correlations among distributions of Ca2+ and V m kinetic functions that regulate SAN cell clock coupling during each AP cycle in different individual, isolated SAN cells not in contact with each other. Our numerical model simulations further extended our perspectives to the molecular scale and demonstrated that many ion currents also behave self-similar across autonomic states. Thus, to ensure rapid flexibility of AP firing rates in response to different types and degrees of autonomic input, nature "did not reinvent molecular wheels within the coupled-clock system of pacemaker cells," but differentially engaged or scaled the kinetics of gears that regulate the rate and rhythm at which the "wheels spin" in a given autonomic input context.

4.
Circ Arrhythm Electrophysiol ; 11(6): e005896, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29880528

RESUMEN

BACKGROUND: Spontaneous firing of sinoatrial node cells (SANCs) is regulated by cAMP-mediated, PKA (protein kinase A)-dependent (cAMP/PKA) local subsarcolemmal Ca2+ releases (LCRs) from RyRs (ryanodine receptors). LCRs occur during diastolic depolarization and activate an inward Na+/Ca2+ exchange current that accelerates diastolic depolarization rate prompting the next action potential. PDEs (phosphodiesterases) regulate cAMP-mediated signaling; PDE3/PDE4 represent major PDE activities in SANC, but how they modulate LCRs and basal spontaneous SANC firing remains unknown. METHODS: Real-time polymerase chain reaction, Western blot, immunostaining, cellular perforated patch clamping, and confocal microscopy were used to elucidate mechanisms of PDE-dependent regulation of cardiac pacemaking. RESULTS: PDE3A, PDE4B, and PDE4D were the major PDE subtypes expressed in rabbit SANC, and PDE3A was colocalized with α-actinin, PDE4D, SERCA (sarcoplasmic reticulum Ca2+ ATP-ase), and PLB (phospholamban) in Z-lines. Inhibition of PDE3 (cilostamide) or PDE4 (rolipram) alone increased spontaneous SANC firing by ≈20% (P<0.05) and ≈5% (P>0.05), respectively, but concurrent PDE3+PDE4 inhibition increased spontaneous firing by ≈45% (P<0.01), indicating synergistic effect. Inhibition of PDE3 or PDE4 alone increased L-type Ca2+ current (ICa,L) by ≈60% (P<0.01) or ≈5% (P>0.05), respectively, and PLB phosphorylation by ≈20% (P>0.05) each, but dual PDE3+PDE4 inhibition increased ICa,L by ≈100% (P<0.01) and PLB phosphorylation by ≈110% (P<0.05). Dual PDE3+PDE4 inhibition increased the LCR number and size (P<0.01) and reduced the SR (sarcoplasmic reticulum) Ca2+ refilling time (P<0.01) and the LCR period (time from action potential-induced Ca2+ transient to subsequent LCR; P<0.01), leading to decrease in spontaneous SANC cycle length (P<0.01). When RyRs were disabled by ryanodine and LCRs ceased, dual PDE3+PDE4 inhibition failed to increase spontaneous SANC firing. CONCLUSIONS: Basal cardiac pacemaker function is regulated by concurrent PDE3+PDE4 activation which operates in a synergistic manner via decrease in cAMP/PKA phosphorylation, suppression of LCR parameters, and prolongation of the LCR period and spontaneous SANC cycle length.


Asunto(s)
Potenciales de Acción , Relojes Biológicos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Frecuencia Cardíaca , Nodo Sinoatrial/enzimología , Potenciales de Acción/efectos de los fármacos , Animales , Señalización del Calcio , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Activación Enzimática , Frecuencia Cardíaca/efectos de los fármacos , Cinética , Inhibidores de Fosfodiesterasa 3/farmacología , Inhibidores de Fosfodiesterasa 4/farmacología , Conejos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Nodo Sinoatrial/citología , Nodo Sinoatrial/efectos de los fármacos
6.
Biophys J ; 114(5): 1176-1189, 2018 03 13.
Artículo en Inglés | MEDLINE | ID: mdl-29539403

RESUMEN

Recent data suggest that cardiac pacemaker cell function is determined by numerous time-, voltage-, and Ca-dependent interactions of cell membrane electrogenic proteins (M-clock) and intracellular Ca cycling proteins (Ca-clock), forming a coupled-clock system. Many aspects of the coupled-clock system, however, remain underexplored. The key players of the system are Ca release channels (ryanodine receptors), generating local Ca releases (LCRs) from sarcoplasmic reticulum, electrogenic Na/Ca exchanger (NCX) current, and L-type Ca current (ICaL). We combined numerical model simulations with experimental simultaneous recordings of action potentials (APs) and Ca to gain further insight into the complex interactions within the system. Our simulations revealed a positive feedback mechanism, dubbed AP ignition, which accelerates the diastolic depolarization (DD) to reach AP threshold. The ignition phase begins when LCRs begin to occur and the magnitude of inward NCX current begins to increase. The NCX current, together with funny current and T-type Ca current accelerates DD, bringing the membrane potential to ICaL activation threshold. During the ignition phase, ICaL-mediated Ca influx generates more LCRs via Ca-induced Ca release that further activates inward NCX current, creating a positive feedback. Simultaneous recordings of membrane potential and confocal Ca images support the model prediction of the positive feedback among LCRs and ICaL, as diastolic LCRs begin to occur below and continue within the voltage range of ICaL activation. The ignition phase onset (identified within the fine DD structure) begins when DD starts to notably accelerate (∼0.15 V/s) above the recording noise. Moreover, the timing of the ignition onset closely predicted the duration of each AP cycle in the basal state, in the presence of autonomic receptor stimulation, and in response to specific inhibition of either the M-clock or Ca-clock, thus indicating general importance of the new coupling mechanism for regulation of the pacemaker cell cycle duration, and ultimately the heart rate.


Asunto(s)
Potenciales de Acción , Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Retroalimentación Fisiológica , Modelos Cardiovasculares , Intercambiador de Sodio-Calcio/metabolismo , Animales , Diástole , Conejos , Retículo Sarcoplasmático/metabolismo
7.
J Mol Cell Cardiol ; 98: 73-82, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27363295

RESUMEN

Constitutive Ca(2+)/calmodulin (CaM)-activation of adenylyl cyclases (ACs) types 1 and 8 in sinoatrial nodal cells (SANC) generates cAMP within lipid-raft-rich microdomains to initiate cAMP-protein kinase A (PKA) signaling, that regulates basal state rhythmic action potential firing of these cells. Mounting evidence in other cell types points to a balance between Ca(2+)-activated counteracting enzymes, ACs and phosphodiesterases (PDEs) within these cells. We hypothesized that the expression and activity of Ca(2+)/CaM-activated PDE Type 1A is higher in SANC than in other cardiac cell types. We found that PDE1A protein expression was 5-fold higher in sinoatrial nodal tissue than in left ventricle, and its mRNA expression was 12-fold greater in the corresponding isolated cells. PDE1 activity (nimodipine-sensitive) accounted for 39% of the total PDE activity in SANC lysates, compared to only 4% in left ventricular cardiomyocytes (LVC). Additionally, total PDE activity in SANC lysates was lowest (10%) in lipid-raft-rich and highest (76%) in lipid-raft-poor fractions (equilibrium sedimentation on a sucrose density gradient). In intact cells PDE1A immunolabeling was not localized to the cell surface membrane (structured illumination microscopy imaging), but located approximately within about 150nm inside of immunolabeling of hyperpolarization-activated cyclic nucleotide-gated potassium channels (HCN4), which reside within lipid-raft-rich microenvironments. In permeabilized SANC, in which surface membrane ion channels are not functional, nimodipine increased spontaneous SR Ca(2+) cycling. PDE1A mRNA silencing in HL-1 cells increased the spontaneous beating rate, reduced the cAMP, and increased cGMP levels in response to IBMX, a broad spectrum PDE inhibitor (detected via fluorescence resonance energy transfer microscopy). We conclude that signaling via cAMP generated by Ca(2+)/CaM-activated AC in SANC lipid raft domains is limited by cAMP degradation by Ca(2+)/CaM-activated PDE1A in non-lipid raft domains. This suggests that local gradients of [Ca(2+)]-CaM or different AC and PDE1A affinity regulate both cAMP production and its degradation, and this balance determines the intensity of Ca(2+)-AC-cAMP-PKA signaling that drives SANC pacemaker function.


Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/genética , Expresión Génica , Sistema de Conducción Cardíaco , Nodo Sinoatrial/citología , Nodo Sinoatrial/metabolismo , Animales , Calcio/metabolismo , Calmodulina/metabolismo , Línea Celular , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Activación Enzimática , Activación del Canal Iónico , Mitocondrias , Modelos Biológicos , Miocitos Cardíacos/metabolismo , Especificidad de Órganos/genética , Conejos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Transducción de Señal
8.
J Mol Cell Cardiol ; 86: 168-78, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26241846

RESUMEN

cAMP-PKA protein kinase is a key nodal signaling pathway that regulates a wide range of heart pacemaker cell functions. These functions are predicted to be involved in regulation of spontaneous action potential (AP) generation of these cells. Here we investigate if the kinetics and stoichiometry of increase in PKA activity match the increase in AP firing rate in response to ß-adrenergic receptor (ß-AR) stimulation or phosphodiesterase (PDE) inhibition, that alters the AP firing rate of heart sinoatrial pacemaker cells. In cultured adult rabbit pacemaker cells infected with an adenovirus expressing the FRET sensor AKAR3, the EC50 in response to graded increases in the intensity of ß-AR stimulation (by Isoproterenol) the magnitude of the increases in PKA activity and the spontaneous AP firing rate were similar (0.4±0.1nM vs. 0.6±0.15nM, respectively). Moreover, the kinetics (t1/2) of the increases in PKA activity and spontaneous AP firing rate in response to ß-AR stimulation or PDE inhibition were tightly linked. We characterized the system rate-limiting biochemical reactions by integrating these experimentally derived data into a mechanistic-computational model. Model simulations predicted that phospholamban phosphorylation is a potent target of the increase in PKA activity that links to increase in spontaneous AP firing rate. In summary, the kinetics and stoichiometry of increases in PKA activity in response to a physiological (ß-AR stimulation) or pharmacological (PDE inhibitor) stimuli match those of changes in the AP firing rate. Thus Ca(2+)-cAMP/PKA-dependent phosphorylation limits the rate and magnitude of increase in spontaneous AP firing rate.


Asunto(s)
Potenciales de Acción/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Receptores Adrenérgicos beta/metabolismo , Nodo Sinoatrial/efectos de los fármacos , Animales , Señalización del Calcio , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Humanos , Cinética , Inhibidores de Fosfodiesterasa/administración & dosificación , Fosforilación/efectos de los fármacos , Conejos , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/patología , Transducción de Señal/efectos de los fármacos , Nodo Sinoatrial/metabolismo , Nodo Sinoatrial/patología
9.
Proteomics ; 15(12): 2066-77, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25914232

RESUMEN

Protein acylation plays a critical role in protein localization and function. Acylation is essential for human immunodeficiency virus 1 (HIV-1) assembly and budding of HIV-1 from the plasma membrane in lipid raft microdomains and is mediated by myristoylation of the Gag polyprotein and the copackaging of the envelope protein is facilitated by colocalization mediated by palmitoylation. Since the viral accessory protein NEF has been shown to alter the substrate specificity of myristoyl transferases, and alter cargo trafficking lipid rafts, we hypothesized that HIV-1 infection may alter protein acylation globally. To test this hypothesis, we labeled HIV-1 infected cells with biomimetics of acyl azides, which are incorporated in a manner analogous to natural acyl-Co-A. A terminal azide group allowed us to use a copper catalyzed click chemistry to conjugate the incorporated modifications to a number of substrates to carry out SDS-PAGE, fluorescence microscopy, and enrichment for LC-MS/MS. Using LC-MS/MS, we identified 103 and 174 proteins from the myristic and palmitic azide enrichments, with 27 and 45 proteins respectively that differentiated HIV-1 infected from uninfected cells. This approach has provided us with important insights into HIV-1 biology and is widely applicable to many virological systems.


Asunto(s)
Acilcoenzima A/metabolismo , Biomimética , Infecciones por VIH/metabolismo , VIH-1/fisiología , Palmitoil Coenzima A/metabolismo , Proteoma/análisis , Proteómica/métodos , Acilación , Aciltransferasas/metabolismo , Células Cultivadas , Cromatografía Liquida , Química Clic , Electroforesis en Gel Bidimensional , Infecciones por VIH/virología , Humanos , Mapas de Interacción de Proteínas , Proteoma/metabolismo , Espectrometría de Masas en Tándem , Proteínas Virales/metabolismo
10.
J Mol Cell Cardiol ; 77: 1-10, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25257916

RESUMEN

Recent evidence indicates that the spontaneous action potential (AP) of isolated sinoatrial node cells (SANCs) is regulated by a system of stochastic mechanisms embodied within two clocks: ryanodine receptors of the "Ca(2+) clock" within the sarcoplasmic reticulum, spontaneously activate during diastole and discharge local Ca(2+) releases (LCRs) beneath the cell surface membrane; clock crosstalk occurs as LCRs activate an inward Na(+)/Ca(2+) exchanger current (INCX), which together with If and decay of K(+) channels prompts the "M clock," the ensemble of sarcolemmal-electrogenic molecules, to generate APs. Prolongation of the average LCR period accompanies prolongation of the average AP beating interval (BI). Moreover, the prolongation of the average AP BI accompanies increased AP BI variability. We hypothesized that both the average AP BI and AP BI variability are dependent upon stochasticity of clock mechanisms reported by the variability of LCR period. We perturbed the coupled-clock system by directly inhibiting the M clock by ivabradine (IVA) or the Ca(2+) clock by cyclopiazonic acid (CPA). When either clock is perturbed by IVA (3, 10 and 30 µM), which has no direct effect on Ca(2+) cycling, or CPA (0.5 and 5 µM), which has no direct effect on the M clock ion channels, the clock system failed to achieve the basal AP BI and both AP BI and AP BI variability increased. The changes in average LCR period and its variability in response to perturbations of the coupled-clock system were correlated with changes in AP beating interval and AP beating interval variability. We conclude that the stochasticity within the coupled-clock system affects and is affected by the AP BI firing rate and rhythm via modulation of the effectiveness of clock coupling.


Asunto(s)
Potenciales de Acción , Nodo Sinoatrial/fisiología , Animales , Benzazepinas/farmacología , Relojes Biológicos , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio , Indoles/farmacología , Ivabradina , Contracción Miocárdica , Conejos , Retículo Sarcoplasmático/metabolismo , Análisis de la Célula Individual , Nodo Sinoatrial/citología , Procesos Estocásticos
11.
Hypertension ; 64(6): 1334-43, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25225208

RESUMEN

Heart rate (HR) variability (HRV; beat-to-beat changes in the R-wave to R-wave interval) has attracted considerable attention during the past 30+ years (PubMed currently lists >17 000 publications). Clinically, a decrease in HRV is correlated to higher morbidity and mortality in diverse conditions, from heart disease to fetal distress. It is usually attributed to fluctuation in cardiac autonomic nerve activity. We calculated HRV parameters from a variety of cardiac preparations (including humans, living animals, Langendorff-perfused heart, and single sinoatrial nodal cell) in diverse species, combining this with data from previously published articles. We show that regardless of conditions, there is a universal exponential decay-like relationship between HRV and HR. Using 2 biophysical models, we develop a theory for this and confirm that HRV is primarily dependent on HR and cannot be used in any simple way to assess autonomic nerve activity to the heart. We suggest that the correlation between a change in HRV and altered morbidity and mortality is substantially attributable to the concurrent change in HR. This calls for re-evaluation of the findings from many articles that have not adjusted properly or at all for HR differences when comparing HRV in multiple circumstances.


Asunto(s)
Sistema Nervioso Autónomo/fisiopatología , Electrocardiografía , Cardiopatías/fisiopatología , Frecuencia Cardíaca/fisiología , Corazón/fisiopatología , Animales , Modelos Animales de Enfermedad , Corazón/inervación , Humanos , Conejos , Ratas
12.
Heart Rhythm ; 11(7): 1210-9, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24713624

RESUMEN

BACKGROUND: A reduction of complexity of heart beating interval variability that is associated with an increased morbidity and mortality in cardiovascular disease states is thought to derive from the balance of sympathetic and parasympathetic neural impulses to the heart. However, rhythmic clocklike behavior intrinsic to pacemaker cells in the sinoatrial node (SAN) drives their beating, even in the absence of autonomic neural input. OBJECTIVE: To test how this rhythmic clocklike behavior intrinsic to pacemaker cells interacts with autonomic impulses to the heart beating interval variability in vivo. METHODS: We analyzed beating interval variability in time and frequency domains and by fractal and entropy analyses: (1) in vivo, when the brain input to the SAN is intact; (2) during autonomic denervation in vivo; (3) in isolated SAN tissue (ie, in which the autonomic neural input is completely absent); (4) in single pacemaker cells isolated from the SAN; and (5) after autonomic receptor stimulation of these cells. RESULTS: Spontaneous beating intervals of pacemaker cells residing in the isolated SAN tissue exhibit fractal-like behavior and have lower approximate entropy compared with those in the intact heart. Isolation of pacemaker cells from SAN tissue, however, leads to a loss in the beating interval order and fractal-like behavior. ß-Adrenergic receptor stimulation of isolated pacemaker cells increases intrinsic clock synchronization, decreases their action potential period, and increases system complexity. CONCLUSIONS: Both the average beating interval in vivo and beating interval complexity are conferred by the combined effects of clock periodicity intrinsic to pacemaker cells and their response to autonomic neural input.


Asunto(s)
Sistema Nervioso Autónomo/fisiología , Relojes Biológicos/fisiología , Frecuencia Cardíaca/fisiología , Miocitos Cardíacos/fisiología , Nodo Sinoatrial/fisiología , Potenciales de Acción , Animales , Conejos
13.
J Clin Trials ; 4(1)2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26251764

RESUMEN

The normal heart beat intervals are neither strictly stationary nor completely random, and continuously shift from one period to another. Decoding the ECG identifies this "hidden" information that imparts inherent complexity to the heart-beating interval time series. Loss of this complexity in cardiovascular disease is manifested as a reduction in heart rate variability (HRV) and this reduction correlates with an increase in both morbidity and mortality. Because HRV measurements are noninvasive and easy to perform, they have emerged as an important tool in cardiology. However, the identities of specific mechanisms that underline the changes in HRV that occur in cardiovascular diseases remain largely unknown. Changes in HRV have mainly been interpreted on a neural basis, ie due to changes in autonomic impulses to the heart: sympathetic activity decreases both the average heart beat interval and HRV, and parasympathetic activity increases both. It has now become clear, however, that the heart rate and HRV are also determined by intrinsic properties of the pacemaker cells that comprise the sinoatrial node, and the responses of these properties to autonomic receptor stimulation. Here we review how changes in the properties of coupled-clock mechanisms intrinsic to pacemaker cells that comprise the sinoatrial node and their impaired response to autonomic receptor stimulation are implicated in the changes of HRV observed in heart diseases.

14.
Am J Physiol Heart Circ Physiol ; 304(11): H1428-38, 2013 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-23604710

RESUMEN

The spontaneous action potential (AP) firing rate of sinoatrial node cells (SANCs) involves high-throughput signaling via Ca(2+)-calmodulin activated adenylyl cyclases (AC), cAMP-mediated protein kinase A (PKA), and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of SR Ca(2+) cycling and surface membrane ion channel proteins. When the throughput of this signaling increases, e.g., in response to ß-adrenergic receptor activation, the resultant increase in spontaneous AP firing rate increases the demand for ATP. We hypothesized that an increase of ATP production to match the increased ATP demand is achieved via a direct effect of increased mitochondrial Ca(2+) (Ca(2+)m) and an indirect effect via enhanced Ca(2+)-cAMP/PKA-CaMKII signaling to mitochondria. To increase ATP demand, single isolated rabbit SANCs were superfused by physiological saline at 35 ± 0.5°C with isoproterenol, or by phosphodiesterase or protein phosphatase inhibition. We measured cytosolic and mitochondrial Ca(2+) and flavoprotein fluorescence in single SANC, and we measured cAMP, ATP, and O2 consumption in SANC suspensions. Although the increase in spontaneous AP firing rate was accompanied by an increase in O2 consumption, the ATP level and flavoprotein fluorescence remained constant, indicating that ATP production had increased. Both Ca(2+)m and cAMP increased concurrently with the increase in AP firing rate. When Ca(2+)m was reduced by Ru360, the increase in spontaneous AP firing rate in response to isoproterenol was reduced by 25%. Thus, both an increase in Ca(2+)m and an increase in Ca(2+) activated cAMP-PKA-CaMKII signaling regulate the increase in ATP supply to meet ATP demand above the basal level.


Asunto(s)
Adenosina Trifosfato/metabolismo , Relojes Biológicos/fisiología , Corazón/fisiología , Miocardio/citología , Miocardio/metabolismo , Animales , Señalización del Calcio/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , Separación Celular , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Citosol/metabolismo , Flavoproteínas/metabolismo , Frecuencia Cardíaca/fisiología , Técnicas In Vitro , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/fisiología , Contracción Miocárdica/fisiología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/fisiología , Consumo de Oxígeno/fisiología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Conejos , Receptores Adrenérgicos beta/fisiología , Frecuencia Respiratoria/fisiología
15.
Sci Signal ; 6(260): ra6, 2013 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-23362239

RESUMEN

The spontaneous beating of the heart is governed by spontaneous firing of sinoatrial node cells, which generate action potentials due to spontaneous depolarization of the membrane potential, or diastolic depolarization. The spontaneous diastolic depolarization rate is determined by spontaneous local submembrane Ca²âº releases through ryanodine receptors (RyRs). We sought to identify specific mechanisms of intrinsic Ca²âº cycling by which sinoatrial node cells, but not ventricular myocytes, generate robust, rhythmic local Ca²âº releases. At similar physiological intracellular Ca²âº concentrations, local Ca²âº releases were large and rhythmic in permeabilized sinoatrial node cells but small and random in permeabilized ventricular myocytes. Furthermore, sinoatrial node cells spontaneously released more Ca²âº from the sarcoplasmic reticulum than did ventricular myocytes, despite comparable sarcoplasmic reticulum Ca²âº content in both cell types. This ability of sinoatrial node cells to generate larger and rhythmic local Ca²âº releases was associated with increased abundance of sarcoplasmic reticulum Ca²âº-ATPase (SERCA), reduced abundance of the SERCA inhibitor phospholamban, and increased Ca²âº-dependent phosphorylation of phospholamban and RyR. The increased phosphorylation of RyR in sinoatrial node cells may facilitate Ca²âº release from the sarcoplasmic reticulum, whereas Ca²âº-dependent increase in phosphorylation of phospholamban relieves its inhibition of SERCA, augmenting the pumping rate of Ca²âº required to support robust, rhythmic local Ca²âº releases. The differences in Ca²âº cycling between sinoatrial node cells and ventricular myocytes provide insights into the regulation of intracellular Ca²âº cycling that drives the automaticity of sinoatrial node cells.


Asunto(s)
Relojes Biológicos/fisiología , Calcio/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Nodo Sinoatrial/metabolismo , Animales , Relojes Biológicos/efectos de los fármacos , Proteínas de Unión al Calcio/farmacología , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Miocardio/citología , Miocardio/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Conejos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , Nodo Sinoatrial/citología
16.
Artículo en Inglés | MEDLINE | ID: mdl-26709383

RESUMEN

The heart rate and rhythm are controlled by complex chaotic neural, chemical and hormonal networks which are not strictly regular, but exhibit fluctuations across multiple time scales. A careful assessment of the heart rate variability (HRV) offers clues to this complexity. A reduction in HRV, specifically in advanced age, is associated with increase in morbidity and mortality. Mechanisms that induce this decrease, however, have not been fully elucidated. The classical literature characterizes changes in HRV as a result of changes in the balance of competing influences of the sympathetic and parasympathetic autonomic impulses delivered to the heart. It has now become clear, however, that the heart rate and HRV are also determined by intrinsic properties of the pacemaker cells that comprise sinoatrial node, and that these properties respond to autonomic receptor stimulation in a non-linear mode. That HRV is determined by both the intrinsic properties of pacemaker cells in the sinoatrial node and the competing influences of the two branches of the autonomic neural input to the cells requires an expansion of our perspective about mechanisms that govern HRV in the normal heart, and how HRV changes with aging in health and in heart diseases.

17.
J Mol Cell Cardiol ; 53(5): 687-94, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22921807

RESUMEN

Freshly isolated adult rabbit sinoatrial node cells (f-SANC) are an excellent model for studies of autonomic signaling, but are not amenable to genetic manipulation. We have developed and characterized a stable cultured rabbit SANC (c-SANC) model that is suitable for genetic manipulation to probe mechanisms of spontaneous action potential (AP) firing. After 48 h in culture, c-SANC generate stable, rhythmic APs at 34±0.5°C, at a rate that is 50% less than f-SANC. In c- vs. f-SANC: AP duration is prolonged; phosphorylation of phospholamban at Ser(16) and type2 ryanodine receptor (RyR2) at Ser(2809) are reduced; and the level of type2 regulator of G-protein signaling (RGS2), that facilitates adenylyl cyclases/cAMP/protein kinase A (PKA) via G(i) inhibition, is substantially reduced. Consistent with the interpretation that cAMP/PKA signaling becomes impaired in c-SANC, acute ß-adrenergic receptor stimulation increases phospholamban and RyR2 phosphorylation, enhances RGS2-labeling density, and accelerates the AP firing rate to the similar maximum in c- and f-SANC. Specific PKA inhibition completely inhibits all ß-adrenergic receptor effects. Adv-RGS2 infection, or pertussis toxin treatment to disable G(i)-signaling, each partially rescues the c-SANC spontaneous AP firing rate. Thus, a G(i)-dependent reduction in PKA-dependent protein phosphorylation, including that of Ca(2+) cycling proteins, reduces the spontaneous AP firing rate of c-SANC, and can be reversed by genetic or pharmacologic manipulation of PKA signaling.


Asunto(s)
Potenciales de Acción , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas RGS/metabolismo , Nodo Sinoatrial/citología , Adenoviridae/genética , Agonistas Adrenérgicos beta/farmacología , Animales , Proteínas de Unión al Calcio/metabolismo , Forma de la Célula , Células Cultivadas , AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Vectores Genéticos , Isoproterenol/farmacología , Toxina del Pertussis/farmacología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional , Proteínas RGS/genética , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Sistemas de Mensajero Secundario , Transfección
18.
PLoS One ; 7(5): e37582, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22666369

RESUMEN

BACKGROUND: Mitochondria dynamically buffer cytosolic Ca(2+) in cardiac ventricular cells and this affects the Ca(2+) load of the sarcoplasmic reticulum (SR). In sinoatrial-node cells (SANC) the SR generates periodic local, subsarcolemmal Ca(2+) releases (LCRs) that depend upon the SR load and are involved in SANC automaticity: LCRs activate an inward Na(+)-Ca(2+) exchange current to accelerate the diastolic depolarization, prompting the ensemble of surface membrane ion channels to generate the next action potential (AP). OBJECTIVE: To determine if mitochondrial Ca(2+) (Ca(2+) (m)), cytosolic Ca(2+) (Ca(2+) (c))-SR-Ca(2+) crosstalk occurs in single rabbit SANC, and how this may relate to SANC normal automaticity. RESULTS: Inhibition of mitochondrial Ca(2+) influx into (Ru360) or Ca(2+) efflux from (CGP-37157) decreased [Ca(2+)](m) to 80 ± 8% control or increased [Ca(2+)](m) to 119 ± 7% control, respectively. Concurrent with inhibition of mitochondrial Ca(2+) influx or efflux, the SR Ca(2+) load, and LCR size, duration, amplitude and period (imaged via confocal linescan) significantly increased or decreased, respectively. Changes in total ensemble LCR Ca(2+) signal were highly correlated with the change in the SR Ca(2+) load (r(2) = 0.97). Changes in the spontaneous AP cycle length (Ru360, 111 ± 1% control; CGP-37157, 89 ± 2% control) in response to changes in [Ca(2+)](m) were predicted by concurrent changes in LCR period (r(2) = 0.84). CONCLUSION: A change in SANC Ca(2+) (m) flux translates into a change in the AP firing rate by effecting changes in Ca(2+) (c) and SR Ca(2+) loading, which affects the characteristics of spontaneous SR Ca(2+) release.


Asunto(s)
Calcio/metabolismo , Mitocondrias/metabolismo , Retículo Sarcoplasmático/metabolismo , Nodo Sinoatrial/citología , Nodo Sinoatrial/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Clonazepam/análogos & derivados , Clonazepam/farmacología , Citosol/efectos de los fármacos , Citosol/metabolismo , Conductividad Eléctrica , Frecuencia Cardíaca/efectos de los fármacos , Ventrículos Cardíacos/citología , Indoles/metabolismo , Magnesio/farmacología , Mitocondrias/efectos de los fármacos , Modelos Biológicos , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Periodicidad , Conejos , Compuestos de Rutenio/farmacología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , Nodo Sinoatrial/efectos de los fármacos , Nodo Sinoatrial/fisiología , Tiazepinas/farmacología
19.
J Proteome Res ; 10(11): 5150-62, 2011 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-21905706

RESUMEN

Lipid raft microdomains, a component of detergent resistant membranes (DRMs), are routinely exploited by pathogens during host-cell entry. Multiple membrane-surface proteins mediate Plasmodium ookinete invasion of the Anopheles midgut, a critical step in the parasite life cycle that is successfully targeted by transmission-blocking vaccines (TBV). Given that lipid rafts are a common feature of host-pathogen interactions, we hypothesized that they promote the partitioning of midgut surface proteins and thus facilitate ookinete invasion. In support of this hypothesis, we found that five of the characterized Anopheles TBV candidates, including the leading Anopheles TBV candidate, AgAPN1, are present in Anopheles gambiae DRMs. Therefore, to extend the repertoire of putative midgut ligands that can be targeted by TBVs, we analyzed midgut DRMs by tandem mass spectrometry. We identified 1452 proteins including several markers of DRMs. Since glycosylphosphotidyl inositol (GPI)-anchored proteins partition to DRMs, we characterized the GPI subproteome of An. gambiae midgut brush-border microvilli and found that 96.9% of the proteins identified in the GPI-anchored fractions were also present in DRMs. Our study vastly expands the number of candidate malarial TBV targets for subsequent analysis by the broader community and provides an inferred role for midgut plasmalemma microdomains in ookinete cell invasion.


Asunto(s)
Anopheles/metabolismo , Sistema Digestivo/metabolismo , Proteínas Ligadas a GPI/metabolismo , Interacciones Huésped-Parásitos , Proteínas de Insectos/metabolismo , Microdominios de Membrana/metabolismo , Plasmodium/fisiología , Animales , Anopheles/parasitología , Colesterol/metabolismo , Sistema Digestivo/citología , Epitelio/metabolismo , Femenino , Proteínas Ligadas a GPI/aislamiento & purificación , Glicosilación , Inmunoglobulinas/química , Proteínas de Insectos/aislamiento & purificación , Microvellosidades/metabolismo , Modelos Biológicos , Estructura Terciaria de Proteína
20.
J Mol Cell Cardiol ; 51(5): 740-8, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21835182

RESUMEN

RATIONALE: In sinoatrial node cells (SANC), Ca(2+) activates adenylate cyclase (AC) to generate a high basal level of cAMP-mediated/protein kinase A (PKA)-dependent phosphorylation of Ca(2+) cycling proteins. These result in spontaneous sarcoplasmic-reticulum (SR) generated rhythmic Ca(2+) oscillations during diastolic depolarization, that not only trigger the surface membrane to generate rhythmic action potentials (APs), but, in a feed-forward manner, also activate AC/PKA signaling. ATP is consumed to pump Ca(2+) to the SR, to produce cAMP, to support contraction and to maintain cell ionic homeostasis. OBJECTIVE: Since feedback mechanisms link ATP-demand to ATP production, we hypothesized that (1) both basal ATP supply and demand in SANC would be Ca(2+)-cAMP/PKA dependent; and (2) due to its feed-forward nature, a decrease in flux through the Ca(2+)-cAMP/PKA signaling axis will reduce the basal ATP production rate. METHODS AND RESULTS: O(2) consumption in spontaneous beating SANC was comparable to ventricular myocytes (VM) stimulated at 3 Hz. Graded reduction of basal Ca(2+)-cAMP/PKA signaling to reduce ATP demand in rabbit SANC produced graded ATP depletion (r(2)=0.96), and reduced O(2) consumption and flavoprotein fluorescence. Neither inhibition of glycolysis, selectively blocking contraction nor specific inhibition of mitochondrial Ca(2+) flux reduced the ATP level. CONCLUSIONS: Feed-forward basal Ca(2+)-cAMP/PKA signaling both consumes ATP to drive spontaneous APs in SANC and is tightly linked to mitochondrial ATP production. Interfering with Ca(2+)-cAMP/PKA signaling not only slows the firing rate and reduces ATP consumption, but also appears to reduce ATP production so that ATP levels fall. This distinctly differs from VM, which lack this feed-forward basal cAMP/PKA signaling, and in which ATP level remains constant when the demand changes.


Asunto(s)
Adenosina Trifosfato/biosíntesis , Señalización del Calcio/fisiología , Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/biosíntesis , Retroalimentación Fisiológica , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Nodo Sinoatrial/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Inhibidores Enzimáticos/farmacología , Glucólisis/efectos de los fármacos , Glucólisis/fisiología , Frecuencia Cardíaca/efectos de los fármacos , Frecuencia Cardíaca/fisiología , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , ATPasas de Translocación de Protón Mitocondriales/genética , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Consumo de Oxígeno , Periodicidad , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Conejos , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Nodo Sinoatrial/citología , Nodo Sinoatrial/efectos de los fármacos , Nodo Sinoatrial/fisiología
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