Publications (18) View all
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Article: The relevance of non-excitable cells for cardiac pacemaker function.
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ABSTRACT: Age-dependent changes in the architecture of the sinus node comprise an increasing ratio between fibroblasts and cardiomyocytes. This change is discussed as a potential mechanism for sinus node disease. The goal of this study was to determine the mechanism through which non-excitable cells influence the spontaneous activity of multicellular cardiomyocyte preparations. Cardiomyocyte monolayers (HL-1 cells) or embryonic stem cell-derived cardiomyocytes were used as two- and three-dimensional cardiac pacemaker models. Spontaneous activity and conduction velocity (theta) were monitored by field potential measurements with microelectrode arrays (MEAs). The influence of fibroblasts (WT-fibs) was determined in heterocellular cultures of different cardiomyocyte and fibroblast ratios. The relevance of heterocellular gap junctional coupling was evaluated by the use of fibroblasts deficient for the expression of Cx43 (Cx43(-/-)-fibs). The beating frequency and of heterocellular cultures depended negatively on the fibroblast concentration. Interspersion of fibroblasts in cardiomyocyte monolayers increased the coefficient of the interbeat interval variability. Whereas Cx43(-/-)-fibs decreased theta significantly less than WT-fibs, their effect on the beating frequency and the beat-to-beat variability seemed largely independent of their ability to establish intercellular coupling. These results suggest that electrically integrated, non-excitable cells modulate the excitability of cardiac pacemaker preparations by two distinct mechanisms, one dependent and the other independent of the heterocellular coupling established. Whereas heterocellular coupling enables the fibroblast to depolarize the cardiomyocytes or to act as a current sink, the mere physical separation of the cardiomyocytes by fibroblasts induces bradycardia through a reduction in frequency entrainment.The Journal of Physiology 01/2008; 585(Pt 2):565-78. · 4.72 Impact Factor -
Article: Pyruvate modulates cardiac sarcoplasmic reticulum Ca2+ release in rats via mitochondria-dependent and -independent mechanisms.
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ABSTRACT: The glycolytic product pyruvate has beneficial effects on cardiac contractile function. The postulated cellular mechanisms underlying the positive inotropic effect of pyruvate, however, are contradictory or have remained elusive. Therefore, we studied the effects of pyruvate on cardiac Ca2+ regulation, intracellular pH (pHi) and flavoprotein oxidation using fluorescence confocal microscopy in intact and permeabilized rat ventricular myocytes and single channel recordings from rat cardiac ryanodine receptors (RyRs) incorporated into planar lipid bilayers. In intact cells extracellular pyruvate (10 mM) elevated diastolic [Ca2+]i, which was due, at least in part, to a concomitant acidification of the cytosol. Furthermore, pyruvate increased the amplitude and slowed the kinetics of the electrically evoked [Ca2+]i transient, and augmented sarcoplasmic reticulum (SR) Ca2+ content. Recording of flavoprotein (FAD) fluorescence indicated that pyruvate caused a reduction of mitochondrial redox potential, which is proportional to an increase of the rate of ATP synthesis. Inhibitors of mitochondrial monocarboxylate transport (alpha-cyano-4-hydroxycinnamate, 0.5 mM), adenine nucleotide translocation (atractyloside, 0.3 mM) and the electron transport chain (cyanide, 4 mM) abolished or attenuated the pyruvate-mediated increase of the amplitude of the [Ca2+]i transient, but did not change the effect of pyruvate on diastolic [Ca2+]i. Results from experiments with permeabilized myocytes indicated a direct correlation between ATP/ADP ratio and SR Ca2+ content. Furthermore, pyruvate (4 mM) reduced the frequency of spontaneous Ca2+ sparks by approximately 50%. Single RyR channel recordings revealed a approximately 60% reduction of the open probability of the channel by pyruvate (1 mM), but no change in conductance. This effect of pyruvate on RyR channel activity was neither Ca2+ nor ATP dependent. Taken together, these findings suggest that, in cardiac tissue, pyruvate has a dual effect on SR Ca2+ release consisting of a direct inhibition of RyR channel activity and elevation of SR Ca2+ content. The latter effect was most probably mediated by an enhanced SR Ca2+ uptake due to an augmentation of mitochondria-dependent ATP synthesis.The Journal of Physiology 09/2003; 550(Pt 3):765-83. · 4.72 Impact Factor -
Article: Ca2+ sparks and cellular distribution of ryanodine receptors in developing cardiomyocytes from rat.
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ABSTRACT: Although abundant ryanodine receptors (RyRs) exist in cardiomyocytes from newborn (NB) rat and despite the maturity of their single-channel properties, the RyR contribution to excitation-contraction (E-C) coupling is minimal. Immature arrangement of RyRs in the Ca(2+) release site of the sarcoplasmic reticulum and/or distant RyRs location from the sarcolemmal Ca(2+) signal could explain this quiescence. Consequently, Ca(2+) sparks and their cellular distribution were studied in NB myocytes and correlated with the formation of dyads and transverse (T) tubules. Ca(2+) sparks were recorded in fluo-4-loaded intact ventricular myocytes acutely dissociated from adult and NB rats (0-9 days old). Sparks were defined/compared in the center and periphery of the cell. Co-immunolocalization of RyRs with dihydropyridine receptors (DHPR) was used to estimate dyad formation, while the development of T tubules was studied using di-8-ANEPPS and diIC12. Our results indicate that in NB cells, Ca(2+) sparks exhibited lower amplitude (1.7+/-0.5 vs. 3.6+/-1.7 F/F(0)), shorter duration (47+/-3.2 vs. 54.1+/-3 ms), and larger width (1.7+/-0.8 vs. 1.2+/-0.4 microm) than in adult. Although no significant changes were observed in the overall frequency, central sparks increased from approximately 60% at 0-1 day to 82% at 7-9 days. While immunolocalization revealed many central release sites at 7-8 days, fluorescence labeling of the plasma membrane showed less abundant internal T tubules. This could imply that although during the first week, release sites emerge forming dyads with DHPR-containing T tubules; some of these T tubules may not be connected to the surface, explaining the RyR quiescence during E-C coupling in NB.Journal of Molecular and Cellular Cardiology 07/2008; 44(6):1032-44. · 5.17 Impact Factor -
Article: T-tubule formation in cardiacmyocytes: two possible mechanisms?
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ABSTRACT: We have followed the differentiation of transverse (T) tubules and of the associations between sarcoplasmic reticulum (SR) and either the plasmalemma (peripheral couplings) or the T tubules (dyads) in postnatal rat ventricular myocytes using electron microscopy. Dyads and peripheral couplings are collectively called Ca(2+) Release Units (CRUs) because they are the sites at which Ca(2+) is released from the SR. Profiles of T tubules, caveolae and dyads are mostly at the cell edge in early postnatal days and are found with increased frequency in the cell interior during the first two postnatal weeks. Using ferritin to trace continuity of T tubules lumen with the extracellular space, we find that some of T tubules (between approximately 6 and 25%), either singly or within dyads, lack ferritin in their lumen. The percentage of tubules that do not contain ferritin decreases slightly during postnatal differentiation and is not very different at the cells' edges and interior. We propose that T tubules form as invaginations of the plasmalemma that penetrate inward driven by accrual of membrane lipids and specific proteins. This occurs by a dual mechanism: either by the independent flow of SR and T tubule proteins into the two separate membranes or by the fusion of preformed vesicle tandems into the dyads. Most of the CRUs (approximately 86%) are constituted by peripheral couplings and ferritin containing dyads, thus constituting CRUs in which Ca(2+ )release from the SR is initiated by a membrane depolarization. In the remaining CRUs, activation of Ca(2+) release must be dependent on some other mechanisms.Journal of Muscle Research and Cell Motility 02/2007; 28(4-5):231-41. · 1.98 Impact Factor -
Article: Cell culture modifies Ca2+ signaling during excitation-contraction coupling in neonate cardiac myocytes.
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ABSTRACT: In heart, the excitation-contraction coupling (ECC) mechanism changes during development. Primary cell culture has been used to study Ca(2+) signaling in newborn (NB) rat heart. In this work, the effects of cell culture on the action potential (AP) and ECC Ca(2+) signaling during development were investigated. Specifically, AP, Ca(2+) currents (I(Ca)), and ryanodine receptor (RyR) properties (i.e. density, distribution, and contribution to Ca(2+) transients and Ca(2+) sparks) were defined in cultured myocytes (CM) from 0-day-old NB rat at different times in culture (1-4 days). Compared with acutely dissociated myocytes (ADM) from NB of equivalent ages (1-4 days), CM showed lower RyR density (50% at 1 day, 25% at 4 days), but larger RyR contribution to the Ca(2+) transient (25% at 1 day, 57% at 4 days). Additionally, Ca(2+) sparks were larger, longer, wider, and more frequent in CM than in ADM. RyR cellular distribution also showed different arrangement. While in CM, RyRs were located peripherally, in ADM of equivalent ages a sarcomeric arrangement was predominant. Finally, CM showed a two-fold increase in sarcolemmal Ca(2+) entry during the AP. These results indicated that primary culture is a feasible model to study Ca(2+) signaling in heart; however, it does not precisely reproduce what occurs in ECC during development.Cell Calcium 02/2007; 41(1):13-25. · 3.77 Impact Factor
