1,721,087 research outputs found
Expression and function of dipeptidyl-aminopeptidase-like protein 6 (DPPX) as a putative b-subunit of human cardiac transient outward current encoded by Kv4.3
No full textDipeptidyl-aminopeptidase-like protein 6 (DPPX) was recently shown in the brain to modulate the kinetics of transient A-type currents by accelerating inactivation and recovery from inactivation. Since the kinetics of human cardiac transient outward current (I(to)) are not mimicked by coexpression of the alpha-subunit Kv4.3 with its known beta-subunit KChIP2, we have tested the hypothesis that DPPX may serve as an additional beta-subunit in the human heart. With quantitative real-time RT-PCR strong mRNA expression of DPPX was detected in human ventricles and was verified at the protein level in human but not in rat heart by a DPPX-specific antibody. Co-expression of DPPX with Kv4.3 in Chinese hamster ovary cells produced I(to)-like currents, but compared with expression of KChIP2a and Kv4.3, the time constant of inactivation was faster, the potential of half-maximum steady-state inactivation was more negative and recovery from inactivation was delayed. Co-expression of DPPX in addition to Kv4.3 and KChIP2a produced similar current kinetics as in human ventricular myocytes. We therefore propose that DPPX is an essential component of the native cardiac I(to) channel complex in human heart
The transmembrane beta-subunits KCNE1, KCNE2, and DPP6 modify pharmacological effects of the antiarrhythmic agent tedisamil on the transient outward current Ito
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Is SAP97 the main modulator of regulating Kv ion channels in dilated cardiomyopathy?
No full textBackground: Dilated cardiomyopathy is a multifactorial disease therefore it can be expected that
several gene expression is altered in the heart. The exact nature of those changes has not been
resolved yet. Voltage-dependent potassium channels regulate membrane excitability and cell communication
between cardiomyocytes. Cardiac ion channels are assembled as a homomeric and heteromeric
tetramers composed of a- and b-subunits providing the molecular basis for the
transmembrane ionic currents. The synapse-associated protein 97 (SAP97) can associate with different
potassium ion channels modulating and anchoring these proteins at the plasma membrane.
Aim and Methods: The aim of this study was to compare the expression of Kv ion channels and
the SAP97 in the cardiomyocytes of failing heart. We investigated the expression of the Kv ion channels
by molecular biological techniques (real-time qPCR and immunoblotting) in the heart of failing
and non-failing human ventricular preparations.
Results: The a-subunits of mRNA of channels did not change significantly or we measured mild upor
down-regulation. HERG and KvLQT1 were slightly up regulated but the KChIP2 gene significantly
decreased 50% as the MIRP4 in the left ventricle tissues of DCM patients comparing to those of
undiseased controls. Immunoblotting studies revealed that the protein expression of these
a-subunits is significantly decreased in the samples of patients.
Conclusion: DCM remodel the expression of delayed rectifier potassium channel genes and proteins.
Down-regulation was significant in the anchoring-subunits and little changed in the pore
forming a-subunits. The decreased SAP97 expression suggests that alterations in regulation of potassium
ion channel expression may play a main role in the development of pathological cardiac
repolarization in cardiomyopathy
Dpp10 - A New Putative Regulatory ß-subunit Of Ito In Failing And Non-failing Human Heart
No full textBackground: Recently we reported that the dipeptidyl-aminopeptidase-like protein DPP6 serves as a regulatory ß-subunit for cardiac Kv4.3 channels. DPP6 is up-regulated in human failing hearts in which Kv4.3 and the ß-subunit KChIP2 are down-regulated. Here, we provide evidence for the presence of a new member of this protein family, i.e. DPP10, in failing and non-failing human hearts and investigate the role of this putative ß-subunit in regulating transient outward current.
Methods: mRNA was extracted from samples of 5 failing and 5 non-failing human hearts and quantified by realtime PCR. Functional interaction of DPP10 and Kv4.3 was studied in co-expression experiments (Chinese hamster ovary CHO cells) with standard voltage clamp techniques.
Results: Expression level of DPP10 was 85±13 fg/ng in failing and 33±6 fg/ng total RNA in non-failing hearts (P<0.01), amounting to a 2.6fold reduction. In comparison, DPP6 was up-regulated 1.3fold (P<0.001). After co-expression of Kv4.3 and DPP10 in CHO cells, channel complexes were verified in the plasma membrane by immunostaining, suggesting proper trafficking as with co-expression of Kv4.3 and KChIP2. Expression of Kv4.3 alone failed to yield Ito current, but robust Ito amplitudes were measured after co-expression of Kv4.3 and DPP10, DDP6, or KChIP2. Compared with the conventional co-expression combination of Kv4.3 and KChIP2, kinetics of Ito inactivation were accelerated in Kv4.3 plus DPP10 channels ({tau}fast: 56± 3 ms and 5.9±0.4 ms, respectively; P<0.01), however, recovery from inactivation was not affected ({tau}rec: 53± 7 ms vs. 58±13 ms). Co-expression of Kv4.3 with KChIP2 plus DPP10 maintained rapid inactivation of Ito ({tau}fast 8±1ms), but enhanced recovery kinetics ({tau}rec: 13±2 ms, P<0.001). The role of glycosylation for channel kinetics was studied with the glycosylation inhibitor tunicamicin (10 μg/ml; 24h). In the presence of tunicamicin, Ito inactivation and recovery were slowed ({tau}fast 51± 4 ms and {tau}rec: 42±3 ms), suggesting importance of glycosylation for channel function. Kinetics were similarly slowed when extracellular domain of DDP10 was deleted ({tau}fast: 48±5 ms, {tau}rec: 30± 4 ms).
Conclusion: DPP10, like DPP6 and KChIP2, contributes to regulation of Ito in normal and diseased human hearts
Accessory subunits alter the temperature sensitivity of Kv4.3 channel complexes.
No full textIn human atrial myocytes the transient outward current I(to) develops a conspicuous faster inactivation with increasing temperatures. Since β-subunits are known to modulate I(to) current kinetics, we hypothesized that the temperature sensitivity of I(to) is not only determined by the property of the ion-passing α-subunit Kv4.3 but also by its interaction with accessory β-subunits. We therefore studied the influence of the transmembrane β-subunits KCNE1, KCNE2 and DPP6 on Kv4.3/KChIP2 channels in CHO cells at room temperature and at physiological temperature. Exposure to 37°C caused a significant acceleration of the channel kinetics, whereas current densities and voltage dependences remained unaltered at 37°C compared to 23°C. However, Kv4.3/KChIP2 channels without transmembrane β-subunits showed the strongest temperature sensitivity with considerably increased rates of activation and inactivation at 37°C. KCNE2 significantly slowed the current kinetics at 37°C compared to Kv4.3/KChIP2 channels, whereas KCNE1 did not influence the channel properties at both temperatures. Interestingly, the accelerating effects of DPP6 on current kinetics described at 23°C were diminished at physiological temperature, thus at 37°C current kinetics became remarkably similar for channel complexes Kv4.3/KChIP2 with and without DPP6 isoforms. A Markov state model was developed on the basis of experimental measurements to simulate the influence of β-subunits on Kv4.3 channel complex at both temperatures. In conclusion, the remarkably fast kinetics of the native I(to) at 37°C could be reproduced by co-expressing Kv4.3, KChIP2, KCNE2 and DPP6 in CHO cells, whereas the high temperature sensitivity of human I(to) could be not mimicked
Cardiac tissue engineering
No full textRecent progress in implantations of differentiated cardiac and non-cardiac cells as well as adult stem cells into the heart suggests that the irreversible loss of viable cardiac myocytes that occurs during myocardial infarction can be at least partly substituted. We evaluated an alternative approach by reconstituting cardiac tissue grafts in vitro and implanting them as spontaneously and coherently contracting tissues. For this purpose we have optimized a method to generate ring-shaped three-dimensional engineered heart tissue (EHT) in vitro from neonatal rat cardiac myocytes. When subjected to isometric force measurements in organ baths, electrically stimulated EHTs exhibit a Frank-Starling behavior, a positive inotropic response to increases in extracellular calcium, a positive inotropic and lusitropic response to isoprenaline, and a negative inotropic response to the muscarinic agonist carbachol ('accentuated antagonism'). Twitch tension under maximal calcium amounts to 1-2 mN/mm(2). Importantly, passive (resting) tension is low, yielding a ratio of active/passive tension of approximately 1.5 under basal and 14 under maximal calcium. Morphologically, EHTs represent a highly interconnected three-dimensional network of cardiac myocytes resembling loose cardiac tissue with a high fraction of binucleated cardiac myocytes, strong eosin staining and elongated centrally located nuclei. Electron microscopy demonstrated well developed sarcomeric structures, T-tubules, SR vesicles, T-tubule-SR-junctions, all types of intercellular connective structures, and a basement membrane. Thus, EHTs comprise functional and morphological properties of intact, ventricular myocardium. First implantation experiments of EHTs in the peritoneum of Fischer 344 rats showed that EHTs survived for at least 14 days, maintained a network of differentiated cardiac myocytes, and were strongly vascularzed. Thus, EHTs may serve as material for a novel tissue replacement approach. (C) 2002 Elsevier Science B.V. All rights reserved
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