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7.
Curr Pharm Des ; 21(39): 5636-42, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26323416

RESUMO

Over the past 30 years, much has been learned about the impact of development on drug disposition (i.e., pharmacokinetics). This is not true concerning drug action (i.e., pharmacodynamics). As a consequence, in clinical therapeutics and the drug development process, assumptions are often times made that a specific systemic drug exposure that is associated with desired drug action in adults will produce the same response in children. A review of the literature would suggest that this assumption may, in some cases, be an errant one. The relative paucity of information concerning developmental pharmacodynamics is associated with the challenge of assessing and quantitating drug response in vivo in infants and children. An approach to overcome these difficulties has been the evolution of biomarkers that are functional in nature in that they are capable of measuring drug response in both a time and age dependent fashion. The purpose of this review is to illustrate how functional biomarkers capable of assessing drug response/ effect in the developing child are developed and being evaluated for their clinical utility.


Assuntos
Biomarcadores/metabolismo , Ensaios Clínicos como Assunto , Pediatria , Farmacocinética , Farmacologia , Criança , Humanos
8.
Biochim Biophys Acta ; 1852(8): 1619-29, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25916634

RESUMO

Alternative splicing (AS) plays an important role in regulating mammalian heart development, but a link between misregulated splicing and congenital heart defects (CHDs) has not been shown. We reported that more than 50% of genes associated with heart development were alternatively spliced in the right ventricle (RV) of infants with tetralogy of Fallot (TOF). Moreover, there was a significant decrease in the level of 12 small cajal body-specific RNAs (scaRNAs) that direct the biochemical modification of specific nucleotides in spliceosomal RNAs. We sought to determine if scaRNA levels influence patterns of AS and heart development. We used primary cells derived from the RV of infants with TOF to show a direct link between scaRNA levels and splice isoforms of several genes that regulate heart development (e.g., GATA4, NOTCH2, DAAM1, DICER1, MBNL1 and MBNL2). In addition, we used antisense morpholinos to knock down the expression of two scaRNAs (scarna1 and snord94) in zebrafish and saw a corresponding disruption of heart development with an accompanying alteration in splice isoforms of cardiac regulatory genes. Based on these combined results, we hypothesize that scaRNA modification of spliceosomal RNAs assists in fine tuning the spliceosome for dynamic selection of mRNA splice isoforms. Our results are consistent with disruption of splicing patterns during early embryonic development leading to insufficient communication between the first and second heart fields, resulting in conotruncal misalignment and TOF. Our findings represent a new paradigm for determining the mechanisms underlying congenital cardiac malformations.


Assuntos
Processamento Alternativo/genética , Corpos Enovelados/genética , Coração/embriologia , Coração/crescimento & desenvolvimento , MicroRNAs/fisiologia , Animais , Animais Geneticamente Modificados , Células Cultivadas , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/genética , Humanos , Lactente , Recém-Nascido , Vertebrados/embriologia , Vertebrados/genética , Vertebrados/crescimento & desenvolvimento , Peixe-Zebra
9.
Sci Transl Med ; 4(154): 154ra135, 2012 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-23035047

RESUMO

Monogenic diseases are frequent causes of neonatal morbidity and mortality, and disease presentations are often undifferentiated at birth. More than 3500 monogenic diseases have been characterized, but clinical testing is available for only some of them and many feature clinical and genetic heterogeneity. Hence, an immense unmet need exists for improved molecular diagnosis in infants. Because disease progression is extremely rapid, albeit heterogeneous, in newborns, molecular diagnoses must occur quickly to be relevant for clinical decision-making. We describe 50-hour differential diagnosis of genetic disorders by whole-genome sequencing (WGS) that features automated bioinformatic analysis and is intended to be a prototype for use in neonatal intensive care units. Retrospective 50-hour WGS identified known molecular diagnoses in two children. Prospective WGS disclosed potential molecular diagnosis of a severe GJB2-related skin disease in one neonate; BRAT1-related lethal neonatal rigidity and multifocal seizure syndrome in another infant; identified BCL9L as a novel, recessive visceral heterotaxy gene (HTX6) in a pedigree; and ruled out known candidate genes in one infant. Sequencing of parents or affected siblings expedited the identification of disease genes in prospective cases. Thus, rapid WGS can potentially broaden and foreshorten differential diagnosis, resulting in fewer empirical treatments and faster progression to genetic and prognostic counseling.


Assuntos
Doenças Genéticas Inatas/genética , Genoma Humano/genética , Unidades de Terapia Intensiva Neonatal , Análise de Sequência de DNA/métodos , Conexina 26 , Conexinas , Humanos , Recém-Nascido , Estudos Retrospectivos
11.
Circ Cardiovasc Genet ; 5(3): 279-86, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22528145

RESUMO

BACKGROUND: The importance of noncoding RNAs (ncRNA), especially microRNAs (miRNAs), for maintaining stability in the developing vertebrate heart has recently become apparent; however, there is little known about the expression pattern of ncRNA in the human heart with developmental anomalies. METHODS AND RESULTS: We examined the expression of miRNAs and small nucleolar RNAs (snoRNAs) in right ventricular myocardium from 16 infants with nonsyndromic tetralogy of Fallot (TOF) without a 22q11.2 deletion, 3 fetal heart samples, and 8 normally developing infants. We found 61 miRNAs and 135 snoRNAs to be significantly changed in expression in myocardium from children with TOF compared with normally developing comparison subjects. The pattern of ncRNA expression in TOF myocardium had a surprising resemblance to expression patterns in fetal myocardium, especially for the snoRNAs. Potential targets of miRNAs with altered expression were enriched for gene networks of importance to cardiac development. We derived a list of 229 genes known to be critical to heart development and found 44 had significantly changed expression in TOF myocardium relative to normally developing myocardium. These 44 genes had significant negative correlation with 33 miRNAs, each of which also had significantly changed expression. The primary function of snoRNAs is targeting specific nucleotides of ribosomal RNAs and spliceosomal RNAs for biochemical modification. The targeted nucleotides of the differentially expressed snoRNAs were concentrated in the 28S and 18S ribosomal RNAs and 2 spliceosomal RNAs, U2 and U6. In addition, in myocardium from children with TOF, we observed splicing variants in 51% of genes that are critical for cardiac development. Taken together, these observations suggest a link between levels of snoRNA that target spliceosomal RNAs, spliceosomal function, and heart development. CONCLUSIONS: This is the first report characterizing ncRNA expression in a congenital heart defect. The striking shift in expression of ncRNAs reflects a fundamental change in cell biology, likely impacting expression, transcript splicing, and translation of developmentally important genes and possibly contributing to the cardiac defect.


Assuntos
Miocárdio/metabolismo , RNA não Traduzido/metabolismo , Tetralogia de Fallot/genética , Pré-Escolar , Análise por Conglomerados , Feminino , Humanos , Lactente , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA não Traduzido/genética
13.
Am J Physiol Heart Circ Physiol ; 293(6): H3471-9, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17933975

RESUMO

The macroscopic volume-regulated anion current (VRAC) is regulated by both intracellular and extracellular ATP, which has important implications in signaling and regulation of cellular excitability. The outwardly rectifying Cl(-) channel (ORCC) is a major contributor to the VRAC. This study investigated the effects of intracellular and extracellular ATP on the ORCCs expressed in the human cardiovascular system. With inside-out single-channel patch-clamp techniques, ORCCs were recorded from myocytes isolated from human atrium and septal ventricle and from primary cells originating from human coronary artery endothelium and human coronary artery smooth muscle. ORCCs from all of these tissues had similar biophysical properties, i.e., they were outwardly rectifying in symmetrical Cl(-) solutions, exhibited a slope conductance of approximately 90-100 pS at positive potentials and approximately 22 pS at negative potentials, and had a high open probability that was independent of voltage or time. The presence of ATP at the cytosolic face of the membrane increased the number of patches that contained functional ORCC but had no effect on gating. In contrast, "extracellular" ATP (in pipette solution) had no effect on the proportion of patches in which ORCC was detected but strongly reduced the open probability by increasing the closed dwell time. The potency order for nucleotides to affect gating was ATPgammaS > ATP = UTP > ADP > AMP, which suggests that a negatively charged phosphate group is involved in ORCC block. Our findings are consistent with a role of ORCC in the human cardiovasculature (atrium, ventricle, and coronary arteries). Regulation of ORCC by extracellular ATP suggests that this channel may have an important role in maintaining electrical activity and membrane potential under conditions in which extracellular ATP levels are elevated, such as with ATP release from nerve endings or during pathophysiological conditions.


Assuntos
Trifosfato de Adenosina/metabolismo , Canais de Cloreto/metabolismo , Vasos Coronários/metabolismo , Citosol/metabolismo , Líquido Extracelular/metabolismo , Ativação do Canal Iônico , Miócitos Cardíacos/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Adolescente , Adulto , Células Cultivadas , Criança , Pré-Escolar , Células Endoteliais/metabolismo , Feminino , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Humanos , Lactente , Masculino , Potenciais da Membrana , Pessoa de Meia-Idade , Miócitos Cardíacos/patologia , Miócitos de Músculo Liso/metabolismo , Técnicas de Patch-Clamp , Fatores de Tempo , Uridina Trifosfato/metabolismo
14.
Am J Physiol Heart Circ Physiol ; 293(5): H2826-35, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17720773

RESUMO

Selective stimulation of beta(2)-adrenergic receptors (ARs) in newborn rabbit ventricular myocardium invokes a positive inotropic effect that is lost during postnatal maturation. The underlying mechanisms for this age-related stimulatory response remain unresolved. We examined the effects of beta(2)-AR stimulation on L-type Ca(2+) current (I(Ca,L)) during postnatal development. I(Ca,L) was measured (37 degrees C; either Ca(2+) or Ba(2+) as the charge carrier) using the whole-cell patch-clamp technique in newborn (1 to 5 days old) and adult rabbit ventricular myocytes. Ca(2+) transients were measured concomitantly by dialyzing the cell with indo-1. Activation of beta(2)-ARs (with either 100 nM zinterol or 1 microM isoproterenol in the presence of the beta(1)-AR antagonist, CGP20712A) stimulated I(Ca,L) twofold in newborns but not in adults. The beta(2)-AR-mediated increase in Ca(2+) transient amplitude in newborns was due exclusively to the augmentation of I(Ca,L). Zinterol increased the rate of inactivation of I(Ca,L) and increased the Ca(2+) flux integral. The beta(2)-AR inverse agonist, ICI-118551 (500 nM), but not the beta(1)-AR antagonist, CGP20712A (500 nM), blocked the response to zinterol. Unexpectedly, the PKA blockers, H-89 (10 microM), PKI 6-22 amide (10 microM), and Rp-cAMP (100 microM), all failed to prevent the response to zinterol but completely blocked responses to selective beta(1)-AR stimulation of I(Ca,L) in newborns. Our results demonstrate that in addition to the conventional beta(1)-AR/cAMP/PKA pathway, newborn rabbit myocardium exhibits a novel beta(2)-AR-mediated, PKA-insensitive pathway that stimulates I(Ca,L). This striking developmental difference plays a major role in the age-related differences in inotropic responses to beta(2)-AR agonists.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2 , Agonistas Adrenérgicos beta/administração & dosagem , Canais de Cálcio Tipo L/fisiologia , Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação do Canal Iônico/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Animais Recém-Nascidos , Canais de Cálcio Tipo L/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação do Canal Iônico/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Coelhos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
15.
Biochem Biophys Res Commun ; 355(2): 338-41, 2007 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-17303081

RESUMO

Phospholemman (PLM) is a small sarcolemmal protein that modulates the activities of Na(+)/K(+)-ATPase and the Na(+)/Ca(2+) exchanger (NCX), thus contributing to the maintenance of intracellular Na(+) and Ca(2+) homeostasis. We characterized the expression and subcellular localization of PLM, NCX, and the Na(+)/K(+)-ATPase alpha1-subunit during perinatal development. Western blotting demonstrates that PLM (15kDa), NCX (120kDa), and Na(+)/K(+)-ATPase alpha-1 (approximately 100kDa) proteins are all more than 2-fold higher in ventricular membrane fractions from newborn rabbit hearts (1-4-day old) compared to adult hearts. Our immunocytochemistry data demonstrate that PLM, NCX, and Na(+)/K(+)-ATPase are all expressed at the sarcolemma of newborn ventricular myocytes. Taken together, our data indicate that PLM, NCX, and Na(+)/K(+)-ATPase alpha-1 proteins have similar developmental expression patterns in rabbit ventricular myocardium. Thus, PLM may have an important regulatory role in maintaining cardiac Na(+) and Ca(2+) homeostasis during perinatal maturation.


Assuntos
Crescimento , Proteínas de Membrana/metabolismo , Miocárdio/metabolismo , Fosfoproteínas/metabolismo , Animais , Animais Recém-Nascidos , Coelhos , Trocador de Sódio e Cálcio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo
16.
Am J Physiol Heart Circ Physiol ; 291(2): H543-51, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16501027

RESUMO

Cardiac ATP-sensitive K+ (K(ATP)) channels are formed by Kir6.2 and SUR2A subunits. We produced transgenic mice that express dominant negative Kir6.x pore-forming subunits (Kir6.1-AAA or Kir6.2-AAA) in cardiac myocytes by driving their expression with the alpha-myosin heavy chain promoter. Weight gain and development after birth of these mice were similar to nontransgenic mice, but an increased mortality was noted after the age of 4-5 mo. Transgenic mice lacked cardiac K(ATP) channel activity as assessed with patch clamp techniques. Consistent with a decreased current density observed at positive voltages, the action potential duration was increased in these mice. Some myocytes developed EADs after isoproterenol treatment. Hemodynamic measurements revealed no significant effects on ventricular function (apart from a slightly elevated heart rate), whereas in vivo electrophysiological recordings revealed a prolonged ventricular effective refractory period in transgenic mice. The transgenic mice tolerated stress less well as evident from treadmill stress tests. The proarrhythmogenic features and lack of adaptation to a stress response in transgenic mice suggest that these features are intrinsic to the myocardium and that K(ATP) channels in the myocardium have an important role in protecting the heart from lethal arrhythmias and adaptation to stress situations.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/fisiologia , Miócitos Cardíacos/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Animais , Western Blotting , Eletrocardiografia , Eletrofisiologia , Ventrículos do Coração/citologia , Hemodinâmica/fisiologia , Canais KATP , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/genética , Pericárdio/fisiologia , Esforço Físico/fisiologia , Regiões Promotoras Genéticas/genética , RNA/biossíntese , RNA/genética , Período Refratário Eletrofisiológico/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcolema/metabolismo , Frações Subcelulares/metabolismo , Função Ventricular
17.
Pediatr Res ; 58(2): 185-92, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16085792

RESUMO

Prevailing data suggest that sarcolemmal ATP-sensitive (K(ATP)) channels in the adult heart consist of Kir6.2 and SUR2A subunits, but the expression of other K(ATP) channel subunits (including SUR1, SUR2B, and Kir6.1) is poorly defined. The situation is even less clear for the immature heart, which shows a remarkable resistance to hypoxia and metabolic stress. The hypoxia-induced action potential shortening and opening of sarcolemmal K(ATP) channels that occurs in adults is less prominent in the immature heart. This might be due in part to the different biophysical and pharmacological properties of K(ATP) channels of immature and adult K(ATP) channels. Because these properties are largely conferred by subunit composition, it is important to examine the relative expression levels of the various K(ATP) channel subunits during maturation. We therefore used RNAse protection assays, reverse transcription-PCR approaches, and Western blotting to characterize the mRNA and protein expression profiles of K(ATP) channel subunits in fetal, neonatal, and adult mouse heart. Our data indicate that each of the K(ATP) channel subunits (Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B) is expressed in the mouse heart at all of the developmental time points studied. However, the expression level of each of the subunits is low in the fetal heart and progressively increases with maturation. Each of the subunits seems to be expressed in ventricular myocytes with a subcellular expression pattern matching that found in the adult. Our data suggest that the K(ATP) channel composition may change during maturation, which has important implications for K(ATP) channel function in the developing heart.


Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Miocárdio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Trifosfato de Adenosina/metabolismo , Processamento Alternativo , Animais , Western Blotting , Linhagem Celular , Membrana Celular/metabolismo , Primers do DNA/química , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Humanos , Imuno-Histoquímica , Canais KATP , Camundongos , Células Musculares/metabolismo , Isoformas de Proteínas , RNA/metabolismo , RNA Mensageiro/metabolismo , Receptores de Droga , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/metabolismo , Receptores de Sulfonilureias , Distribuição Tecidual , Transfecção , Regulação para Cima
18.
BMC Physiol ; 5(1): 1, 2005 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-15647111

RESUMO

BACKGROUND: Electrophysiological data suggest that cardiac KATP channels consist of Kir6.2 and SUR2A subunits, but the distribution of these (and other KATP channel subunits) is poorly defined. We examined the localization of each of the KATP channel subunits in the mouse and rat heart. RESULTS: Immunohistochemistry of cardiac cryosections demonstrate Kir6.1 protein to be expressed in ventricular myocytes, as well as in the smooth muscle and endothelial cells of coronary resistance vessels. Endothelial capillaries also stained positive for Kir6.1 protein. Kir6.2 protein expression was found predominantly in ventricular myocytes and also in endothelial cells, but not in smooth muscle cells. SUR1 subunits are strongly expressed at the sarcolemmal surface of ventricular myocytes (but not in the coronary vasculature), whereas SUR2 protein was found to be localized predominantly in cardiac myocytes and coronary vessels (mostly in smaller vessels). Immunocytochemistry of isolated ventricular myocytes shows co-localization of Kir6.2 and SUR2 proteins in a striated sarcomeric pattern, suggesting t-tubular expression of these proteins. Both Kir6.1 and SUR1 subunits were found to express strongly at the sarcolemma. The role(s) of these subunits in cardiomyocytes remain to be defined and may require a reassessment of the molecular nature of ventricular KATP channels. CONCLUSIONS: Collectively, our data demonstrate unique cellular and subcellular KATP channel subunit expression patterns in the heart. These results suggest distinct roles for KATP channel subunits in diverse cardiac structures.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Vasos Coronários/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Animais , Endotélio Vascular/metabolismo , Ventrículos do Coração , Imuno-Histoquímica , Técnicas In Vitro , Canais KATP , Camundongos , Mitocôndrias Cardíacas/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Ratos , Receptores de Droga , Frações Subcelulares/metabolismo , Receptores de Sulfonilureias , Distribuição Tecidual
19.
J Cardiovasc Pharmacol ; 45(2): 165-74, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15654266

RESUMO

The Na+-Ca2+ exchanger (NCX) is up-regulated in the neonatal rabbit heart. Because the duration of membrane depolarization is an important determinant of calcium entry via NCX, pharmacological agents that lengthen the action potential (AP) may significantly increase the amount of activator calcium in newborns. We tested this potentially novel therapeutic strategy by using action potential voltage clamp steps or using dofetilide, a blocker of IKr, to prolong the action potential duration (APD). The effects of changing APD on calcium transients were determined in ventricular myocytes at different developmental stages: newborn (1-4 days), juvenile (9-10 days), and adult ventricular myocytes (35 degrees C; 1 Hz). Calcium transient amplitude in neonatal myocytes increased substantially with clamping with longer APs. In contrast, exposure to dofetilide (0.1, 1, and 10 microM) under current clamp conditions increased APD in a concentration-dependent manner but had no significant effect on calcium transient amplitude in either neonates or adults. When the AP was held constant under voltage clamp conditions, dofetilide decreased the calcium transient amplitude in neonates. This effect is likely related to inhibition of sodium-calcium exchanger and L-type Ca2+ currents (ICa), as observed in separate experiments. These results suggest that dofetilide has a paradoxical effect on APD and calcium transients in the newborn heart.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Antiarrítmicos/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Fenetilaminas/farmacologia , Sulfonamidas/farmacologia , Animais , Animais Recém-Nascidos , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Cardiotônicos/farmacologia , Relação Dose-Resposta a Droga , Feminino , Ventrículos do Coração/citologia , Técnicas In Vitro , Masculino , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-Clamp , Coelhos , Trocador de Sódio e Cálcio/metabolismo
20.
Pediatr Res ; 57(3): 399-403, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15611349

RESUMO

Contractions in neonatal rabbit ventricular myocytes seem to depend predominantly on Ca2+ influx through the Na+-Ca2+ exchanger (NCX). Unexpectedly, neonates are sensitive to the negative inotropic effect of L-type Ca2+ channel blockers. L-type Ca2+ channel blockers depress contractile function indirectly in neonatal myocytes by shortening the action potential duration (APD), thereby decreasing the influx of activator Ca2+ through the NCX. Freshly isolated ventricular myocytes from adult and neonatal (1-5 d) rabbits were electrically stimulated (0.5 Hz; 35 degrees C) while action potential (AP) and Ca2+ transients (Indo-1) were recorded in the absence and presence of nifedipine (10 microM). In separate experiments, cells were voltage-clamped with a constant AP waveform (APD90=170 ms) to determine the effect of nifedipine on Ca2+ transients independent from effects on the AP. Voltage-clamp experiments confirmed that nifedipine blocks L-type Ca2+ current in neonatal myocytes. Nifedipine markedly reduced Ca2+ transient amplitude and APD in both adults (transient=20 +/- 7%; APD90=31 +/- 4% of control) and neonates (transient=38 +/- 10%; APD90=57 +/- 6% of control). When the AP was held constant by voltage clamping, nifedipine significantly reduced the amplitude of Ca2+ transients in adults (27 +/- 9% of control) but had no effect on Ca2+ transient amplitude in neonatal myocytes. These results are consistent with the concept that immature ventricular myocytes are less reliant on L-type calcium channels and are more dependent on NCX for contraction. The negative inotropic effect of L-type Ca2+ channel blockers in neonates is attributable to shortening of the AP.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Nifedipino/farmacologia , Animais , Canais de Cálcio Tipo L/metabolismo , Células Cultivadas , Feminino , Masculino , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Coelhos
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