Biomedical subjects

Leif Hove-Madsen

Publications and source records attributed to Leif Hove-Madsen.

11 recordsLinked to original sources

PubMed2006

The proarrhythmic antihistaminic drug terfenadine increases spontaneous calcium release in human atrial myocytes.

Spontaneous calcium release from the sarcoplasmic reticulum in cardiac myocytes plays a central role in cardiac arrhythmogenesis. Compounds intended for therapeutical use that interfere with intracellular calcium handling may therefore have an undesired proarrhythmic potential. Here we have used isolated human atrial myocytes to compare the effect of the proarrhythmic antihistaminic drug terfenadine with the non-proarrhythmic antihistaminic drugs fexofenadine and rupatadine on intracellular calcium homeostasis. Perforated patch-clamp technique was used to measure ionic currents and to detect spontaneous calcium release from the sarcoplasmic reticulum. Our results show that the compound terfenadine, with known arrhythmogenic effects, inhibits L-type calcium current (I(Ca)) with an IC(50) of 185 nM when cells are stimulated at 1.0 Hz. The inhibitory effect of 0.3 muM terfenadine increased from 19+/-4% at stimulation frequency of 0.2 Hz to 63+/-6% at 2.0 Hz. Moreover, terfenadine also increased spontaneous calcium release from the sarcoplasmic reticulum. At a concentration of 1 muM, terfenadine significantly increased the spontaneous Na-Ca exchange current (I(NCX)) frequency from 0.48+/-0.25 to 1.93+/-0.67 s(-1). In contrast, fexofenadine and rupatadine did not change I(Ca) or the frequency of spontaneous I(NCX). We conclude that the proarrhythmic antihistaminic drug terfenadine alters intracellular calcium handling in isolated human atrial myocytes. This experimental model may be suitable to screen for potential arrhythmogenic side-effects of compounds intended for therapeutical use.

Arrhythmias, Cardiac↗

PubMed2006

Modulation of membrane potential by an acetylcholine-activated potassium current in trout atrial myocytes.

Application of the current-clamp technique in rainbow trout atrial myocytes has yielded resting membrane potentials that are incompatible with normal atrial function. To investigate this paradox, we recorded the whole membrane current (I(m)) and compared membrane potentials recorded in isolated cardiac myocytes and multicellular preparations. Atrial tissue and ventricular myocytes had stable resting potentials of -87 +/- 2 mV and -83.9 +/- 0.4 mV, respectively. In contrast, 50 out of 59 atrial myocytes had unstable depolarized membrane potentials that were sensitive to the holding current. We hypothesized that this is at least partly due to a small slope conductance of I(m) around the resting membrane potential in atrial myocytes. In accordance with this hypothesis, the slope conductance of I(m) was about sevenfold smaller in atrial than in ventricular myocytes. Interestingly, ACh increased I(m) at -120 mV from 4.3 pA/pF to 27 pA/pF with an EC(50) of 45 nM in atrial myocytes. Moreover, 3 nM ACh increased the slope conductance of I(m) fourfold, shifted its reversal potential from -78 +/- 3 to -84 +/- 3 mV, and stabilized the resting membrane potential at -92 +/- 4 mV. ACh also shortened the action potential in both atrial myocytes and tissue, and this effect was antagonized by atropine. When applied alone, atropine prolonged the action potential in atrial tissue but had no effect on membrane potential, action potential, or I(m) in isolated atrial myocytes. This suggests that ACh-mediated activation of an inwardly rectifying K(+) current can modulate the membrane potential in the trout atrial myocytes and stabilize the resting membrane potential.

Acetylcholine↗

PubMed2006

Adenosine A2A receptors are expressed in human atrial myocytes and modulate spontaneous sarcoplasmic reticulum calcium release.

BACKGROUND: Alterations in the cyclic AMP-dependent regulation of the cardiac ryanodine receptor (RyR2) have been proposed to account for increased spontaneous calcium release from the sarcoplasmic reticulum (SR) in patients with heart failure, ventricular tachyarrhythmias and atrial fibrillation. While the adenosine A(2A) receptor (A(2A)R) is known to regulate cyclic AMP levels, expression and function of this receptor in human cardiac myocytes has not been investigated. METHODS: PCR, western blotting and immunofluorescence were used to identify the A(2A)R, and functional effects of A(2A)R stimulation were measured with confocal calcium imaging and patch-clamp technique. RESULTS: The A(2A)R is expressed in the human right atrium and distributed in a banded pattern along the Z-lines, overlapping with the ryanodine receptor. A(2A)R stimulation caused a protein kinase A dependent increase in spontaneous SR calcium release in isolated human atrial myocytes. The A(2A)R agonist CGS21680 increased the frequency of calcium sparks from 0.12+/-0.03 to 0.31+/-0.08 sparks.mum min(-1) (p<0.05) and calcium waves from 0.65+/-0.31 to 5.11+/-1.84 waves.min(-1) (p<0.03). Moreover, spontaneous Na-Ca exchange currents (I(NCX)) increased from 1.19+/-0.17 to 2.50+/-0.42 min(-1) (p<0.001). In contrast, CGS21680 did not alter caffeine inducible calcium release (6.98+/-0.52 vs. 6.82+/-0.57 amol pF(-1), p=0.6) or the spontaneous I(NCX) amplitude (0.32+/-0.05 vs. 0.29+/-0.04 pA pF(-1), p=0.2). Current-voltage relationship and amplitude of the L-type calcium current (1.62+/-0.18 vs. 1.80+/-0.18 pA pF(-1)) were not altered, but calcium release dependent inactivation was faster with CGS21680 (13.4+/-0.7 vs. 15.8+/-1.0 ms, p<0.001). CONCLUSIONS: Adenosine A(2A) receptors are expressed in the human atrial myocardium and modulate the frequency of spontaneous calcium release from the SR.

Adenosine↗

PubMed2006

Effect of aging on the pluripotential capacity of human CD105+ mesenchymal stem cells.

BACKGROUND: Whether aging modifies mesenchymal stem cell (MSC) properties is unknown. AIM: To compare the differentiation capacity of human CD105(+) MSCs obtained from young and elderly donors. METHODS AND RESULTS: Cells were obtained from young (n=10, 24+/-6.4 years) and elderly (n=9, 77+/-8.4 years) donors. Cell senescence was assessed by telomere length assays and lipofuscin accumulation. Cell pluripotentiality was analysed by adipogenic and osteogenic induction media, and myocyte phenotype was attempted with 5-azacytidine (5-AZ). Immunofluorescence, Western blot, transmission electron microscopy and fluo-4 confocal imaging were used to analyse the sarcomere, gap junctions and Ca(2+) dynamics. Cells obtained from young and elderly donors showed no significant differences in relative telomere length (40.1+/-6.4% and 40.3+/-3.6%, p=0.9) and lipofuscin accumulation. Adipogenic and osteogenic potential of CD105(+) MSCs was demonstrated. 5-AZ induced increased expression of sarcomeric proteins without complete sarcomere organization. Treated cells also showed increased presence of connexin-43 both in young and old donor-derived cells. Intercellular communications were verified by the observation of gap junctions and passage of Ca(2+) between neighbouring cells. Spontaneous Ca(2+) raises did not significantly increase after 5-AZ treatment in both age groups. CONCLUSION: Age does not influence the adipogenic and myogenic differentiation potential of human CD105(+) MSCs.

Adult↗

PubMed2006

Store-operated Ca2+ entry modulates sarcoplasmic reticulum Ca2+ loading in neonatal rabbit cardiac ventricular myocytes.

Store-operated Ca2+ entry (SOCE), which is Ca2+ entry triggered by the depletion of intracellular Ca2+ stores, has been observed in many cell types, but only recently has it been suggested to occur in cardiomyocytes. In the present study, we have demonstrated SOCE-dependent sarcoplasmic reticulum (SR) Ca2+ loading (load(SR)) that was not altered by inhibition of L-type Ca2+ channels, reverse mode Na+/Ca2+ exchange (NCX), or nonselective cation channels. In contrast, lowering the extracellular [Ca2+] to 0 mM or adding either 0.5 mM Zn2+ or the putative store-operated channel (SOC) inhibitor SKF-96365 (100 microM) inhibited load(SR) at rest. Interestingly, inhibition of forward mode NCX with 30 microM KB-R7943 stimulated SOCE significantly and resulted in enhanced load(SR). In addition, manipulation of the extracellular and intracellular Na+ concentrations further demonstrated the modulatory role of NCX in SOCE-mediated SR Ca2+ loading. Although there is little knowledge of SOCE in cardiomyocytes, the present results suggest that this mechanism, together with NCX, may play an important role in SR Ca2+ homeostasis. The data reported herein also imply the presence of microdomains unique to the neonatal cardiomyocyte. These findings may be of particular importance during open heart surgery in neonates, in which uncontrolled SOCE could lead to SR Ca2+ overload and arrhythmogenesis.

Age Factors↗

PubMed2005

L-type Ca2+ channel function and expression in neonatal rabbit ventricular myocytes.

L-type Ca(2+) channel-mediated, Ca(2+)-induced Ca(2+) release (CICR) is the dominant mode of excitation-contraction (E-C) coupling in the mature mammalian myocardium but is thought to be absent in the fetal and newborn mammalian myocardium. Furthermore, the characteristics and contributors of E-C coupling at the earliest developmental stages are poorly understood. In this study, we measured [(3)H](+)PN200-110 dihydropyridine binding capacity, functionality and expression of the L-type Ca(2+) channel, and cytosolic [Ca(2+)] ([Ca(2+)](i)) at various developmental stages (3, 6, 10, 20, and 56 days old) to characterize ontogenetic changes in E-C coupling. We found that 1) the whole cell L-type Ca(2+) channel peak current (I(Ca)) density increased slightly in parallel with cell growth, but the current-voltage relationship, the steady-state activation, and the maximum DHP binding and binding affinity did not exhibit significant developmental changes; 2) sarcoplasmic reticulum Ca(2+) dependence of inactivation rates of L-type Ca(2+) channel and peak of I(Ca) density were only observed after 10 days of age, which temporally coincides with transverse (T)-tubule formation; 3) the relationship between [Ca(2+)](i) and voltage changed from a linear relationship at the earliest developmental stages to a "bell-shaped" relationship at the later developmental stages, presumably corresponding to a switch from reverse-mode Na/Ca exchange-dependent to I(Ca)-dependent E-C coupling; and 4) the expression of two different splice variants of Ca(V)1.2, IVS3A and IVS3B, switched from predominantly IVS3A at the earliest stages to IVS3B at the later developmental stages. Our data suggest that whereas the density of functional dihydropyridine receptors (DHPRs) increases only slightly during ontogeny, the enhancement of functional coupling between DHPR and ryanodine receptor is dramatic between the second and third weeks after birth. Furthermore, we found that the differential expression of splice variants during development temporally correlated with the appearance of I(Ca)-dependent E-C coupling and T-tubule formation.

Aging↗

PubMed2005

Characterization of a C3a receptor in rainbow trout and Xenopus: the first identification of C3a receptors in nonmammalian species.

Virtually nothing is known about the structure, function, and evolutionary origins of the C3aR in nonmammalian species. Because C3aR and C5aR are thought to have arisen from the same common ancestor, the recent characterization of a C5aR in teleost fish implied the presence of a C3aR in this animal group. In this study we report the cloning of a trout cDNA encoding a 364-aa molecule (TC3aR) that shows a high degree of sequence homology and a strong phylogenetic relationship with mammalian C3aRs. Northern blotting demonstrated that TC3aR was expressed primarily in blood leukocytes. Flow cytometric analysis and immunofluorescence microscopy showed that Abs raised against TC3aR stained to a high degree all blood B lymphocytes and, to a lesser extent, all granulocytes. More importantly, these Abs inhibited trout C3a-mediated intracellular calcium mobilization in trout leukocytes. A fascinating structural feature of TC3aR is the lack of a significant portion of the second extracellular loop (ECL2). In all C3aR molecules characterized to date, the ECL2 is exceptionally large when compared with the same region of C5aR. However, the exact function of the extra portion of ECL2 is unknown. The lack of this segment in TC3aR suggests that the extra piece of ECL2 was not necessary for the interaction of the ancestral C3aR with its ligand. Our findings represent the first C3aR characterized in nonmammalian species and support the hypothesis that if C3aR and C5aR diverged from a common ancestor, this event occurred before the emergence of teleost fish.

Amino Acid Sequence↗

PubMed2004

Na+/Ca2+ exchange activity in neonatal rabbit ventricular myocytes.

Much less is known about the contributions of the Na(+)/Ca(2+) exchanger (NCX) and sarcoplasmic reticulum (SR) Ca(2+) pump to cell relaxation in neonatal compared with adult mammalian ventricular myocytes. Based on both biochemical and molecular studies, there is evidence of a much higher density of NCX at birth that subsequently decreases during the next 2 wk of development. It has been hypothesized, therefore, that NCX plays a relatively more important role for cytosolic Ca(2+) decline in neonates as well as, perhaps, a role in excitation-contraction coupling in reverse mode. We isolated neonatal ventricular myocytes from rabbits in four different age groups: 3, 6, 10, and 20 days of age. Using an amphotericin-perforated patch-clamp technique in fluo-3-loaded myocytes, we measured the caffeine-induced inward NCX current (I(NCX)) and the Ca(2+) transient. We found that the integral of I(NCX), an indicator of SR Ca(2+) content, was greatest in myocytes from younger age groups when normalized by cell surface area and that it decreased with age. The velocity of Ca(2+) extrusion by NCX (V(NCX)) was linear with [Ca(2+)] and did not indicate saturation kinetics until [Ca(2+)] reached 1-3 microM for each age group. There was a significantly greater time delay between the peaks of I(NCX) and the Ca(2+) transient in myocytes from the youngest age groups. This observation could be related to structural differences in the subsarcolemmal microdomains as a function of age.

Animals↗

PubMed2004

Atrial fibrillation is associated with increased spontaneous calcium release from the sarcoplasmic reticulum in human atrial myocytes.

BACKGROUND: Spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) can generate afterdepolarizations, and these have the potential to initiate arrhythmias. Therefore, an association may exist between spontaneous SR Ca2+ release and initiation of atrial fibrillation (AF), but this has not yet been reported. METHODS AND RESULTS: Spontaneous Ca2+ release from the SR, manifested as Ca2+ sparks and Ca2+ waves, was recorded with confocal microscopy in atrial myocytes isolated from patients with and those without AF. In addition, the spontaneous inward current associated with Ca2+ waves was measured with the use of the perforated patch-clamp technique. The Ca2+ spark frequency was higher in 8 patients with AF than in 16 patients without (6.0+/-1.2 versus 2.8+/-0.8 sparks/mm per second, P<0.05). Similarly, the spontaneous Ca2+ wave frequency was greater in patients with AF (2.8+/-0.5 versus 1.1+/-0.3 waves/mm per second, P<0.01). The spontaneous inward current frequency was also higher in 10 patients with AF than in 13 patients without this arrhythmia (0.101+/-0.028 versus 0.031+/-0.007 per second, P<0.05, at a clamped potential of -80 mV). In contrast, both the Ca2+ released from the SR and the Na+-Ca2+ exchange rate induced by a rapid caffeine application were comparable in patients with and without AF. CONCLUSIONS: The observed increase in spontaneous Ca2+ release in patients with AF probably is due to an upregulation of the SR Ca2+ release channel activity, which may contribute to the development of AF.

Aged↗

PubMed2004

Effect of beta-adrenergic stimulation on the relationship between membrane potential, intracellular [Ca2+] and sarcoplasmic reticulum Ca2+ uptake in rainbow trout atrial myocytes.

Long depolarizations cause a steady tonic contraction and induce sarcoplasmic reticulum (SR) Ca(2+)-uptake in trout atrial myocytes. Simultaneous measurements of cytosolic [Ca2+] ([Ca2+]i) and whole membrane current showed an elevated [Ca2+]i throughout the depolarization. Rapid caffeine (Caf) applications at -80 mV before and after a long depolarization were used to determine SR Ca2+ loading and its dependency on membrane potential and [Ca2+]i during depolarization. Following a 10 s depolarization, the maximal SR Ca2+ load was 597 micromol l(-1) and loading was half-maximal at -12 mV. The beta-adrenergic agonist isoproterenol (ISO) did not affect the maximal SR Ca2+ loading but shifted the potential for half-maximal loading by -26 mV. Following a 3 s depolarization, the maximal SR Ca2+ uptake rate (Vmax) was 418 micromol l(-1) s(-1) in control conditions. ISO did not affect Vmax, but significantly lowered the average free Ca2+ transient during the depolarization and shifted the K(0.5) for the relationship between SR Ca2+ uptake and [Ca2+]i from 1.27 in control to 0.8 micromol l(-1) with ISO. Following repetitive 200 ms depolarizations, ISO increased the L-type Ca2+ current (ICa) amplitude by 91+/-29% and the peak Ca2+ transient by 41+/-10%, and decreased the half life of the Ca2+ transient from 151+/-12 to 111+/-6 ms. Using the relationship between [Ca2+]i and SR Ca2+ uptake to calculate the total SR Ca2+ uptake during a Ca2+ transient elicited by a 200 ms depolarization, a significant increase in the SR Ca2+ uptake from 37+/-6 micromol l(-1) in control to 68+/-4 micromol l(-1) with ISO was seen. When normalized to the total Ca2+ transport the contribution of the SR was not significantly different in the absence (35+/-6%) or presence of ISO (41+/-4%). Exposure of cells to ISO and low extracellular [Ca2+] increased ICa by 67+/-40% (N=5) but significantly reduced SR Ca2+ uptake at membrane potentials above -30 mV. Together, these results suggest that (i) ISO has a stimulatory effect on the SR Ca2+ pump that may contribute to the faster decay of the Ca2+ transient, and (ii) the relative contribution of the SR to the Ca2+ removal during relaxation is not altered by ISO in trout atrial myocytes.

Adrenergic beta-Agonists↗

PubMed2003

Triggering of sarcoplasmic reticulum Ca2+ release and contraction by reverse mode Na+/Ca2+ exchange in trout atrial myocytes.

Whole cell patch clamp and intracellular Ca(2+) transients in trout atrial cardiomyocytes were used to quantify calcium release from the sarcoplasmic reticulum (SR) and examine its dependency on the Ca(2+) trigger source. Short depolarization pulses (2-20 ms) elicited large caffeine-sensitive tail currents. The Ca(2+) carried by the caffeine-sensitive tail current after a 2-ms depolarization was 0.56 amol Ca(2+)/pF, giving an SR Ca(2+) release rate of 279 amol Ca(2+). pF(-1). s(-1) or 4.3 mM/s. Depolarizing cells for 10 ms to different membrane potentials resulted in a local maximum of SR Ca(2+) release, intracellular Ca(2+) transient, and cell shortening at 10 mV. Although 100 microM CdCl(2) abolished this local maximum, it had no effect on SR Ca(2+) release elicited by a depolarization to 110 or 150 mV, and the SR Ca(2+) release was proportional to the membrane potential in the range -50 to 150 mV with 100 microM CdCl(2). Increasing the intracellular Na(+) concentration ([Na(+)]) from 10 to 16 mM enhanced SR Ca(2+) release but reduced cell shortening at all membrane potentials examined. In the absence of TTX, SR Ca(2+) release was potentiated with 16 mM but not 10 mM pipette [Na(+)]. Comparison of the total sarcolemmal Ca(2+) entry and the Ca(2+) released from the SR gave a gain factor of 18.6 +/- 7.7. Nifedipine (Nif) at 10 microM inhibited L-type Ca(2+) current (I(Ca)) and reduced the time integral of the tail current by 61%. The gain of the Nif-sensitive SR Ca(2+) release was 16.0 +/- 4.7. A 2-ms depolarization still elicited a contraction in the presence of Nif that was abolished by addition of 10 mM NiCl(2). The gain of the Nif-insensitive but NiCl(2)-sensitive SR Ca(2+) release was 14.8 +/- 7.1. Thus both reverse-mode Na(+)/Ca(2+) exchange (NCX) and I(Ca) can elicit Ca(2+) release from the SR, but I(Ca) is more efficient than reverse-mode NCX in activating contraction. This difference may be due to extrusion of a larger fraction of the Ca(2+) released from the SR by reverse-mode NCX rather than a smaller gain for NCX-induced Ca(2+) release.

Animals↗