Abstract: The alpha(1)-adrenoceptor as well as the AT(1)- and the ET(A)-receptor couple to G-proteins of the Galpha(q/11) family and contribute to the regulation of the transient outward K(+) current (I(to,f)) under pathological conditions such as cardiac hypertrophy or failure. This suggests an important role of Galpha(q/11)-signalling in the physiological regulation of I(to,f). Here, we investigate mice deficient of the Galpha(11) protein (gna11(-/-)) to clarify the physiological role of Galpha(11) signalling in cardiac ion channel regulation. Myocytes from endocardial and epicardial layers were isolated from the left ventricular free wall and investigated using the ruptured-patch whole-cell patch-clamp technique. At +40 mV, epicardial myocytes from gna11(-/-) mice displayed a 23% larger I(to,f) than controls (52.6 + or - 4.1 pApF(-1), n = 20 vs 42.7 + or - 2.8 pApF(-1), n = 26, p < 0.05). Endocardial I(to,f) was similar in gna11(-/-) mice and controls. With the except of minor changes in endocardial myocytes, I(to,f) kinetics were similar in both groups. In the epicardial layer, western blot analysis revealed a 19% higher expression of the K(+)-channel alpha-subunit Kv4.2 in gna11(-/-) mice than in wild type (wt; p < 0.05). The beta-subunit KChIP2b was upregulated by 102% in epicardial myocytes of gna11(-/-) mice (p < 0.01, n = 4). Consistent with the difference in I(to,f), action potential duration was shorter in epicardial cells of gna11(-/-) mice than in wt (p < 0.05), while no difference was found in endocardial myocytes. These results suggest that Galpha(11)-coupled signalling is a central pathway in the regulation of I(to,f). It physiologically exerts a tonic inhibitory influence on the expression of I(to,f) and thereby contributes to the regulation of cardiac repolarisation.
Abstract: Anti-malarial drugs may have severe adverse cardiac effects as a result of their ion channel blocking properties. Here we investigate the effect of the aminoquinolines primaquine and chloroquine on the fast transient outward K(+) current (I(to)) of single epicardial myocytes isolated from the left ventricular free wall of female Wistar rats. The ruptured-patch whole-cell configuration of the patch-clamp technique was used to investigate I(to). At +60 mV, primaquine blocked I(to) amplitude (defined as the current inactivating during a test pulse of 600 ms duration) with an IC(50) of 118±8 μM. I(to) charge was blocked with an IC(50) of 33±2 μM (n=42), indicating open channel block. Chloroquine blocked I(to) amplitude with an IC(50) of 4.6±0.9 mM, while the IC(50) for I(to) charge was 439±63 μM (n=23). The kinetic analysis of the onset of block revealed K(d) values of 52±8 μM (n=18) and 520±60μM (n=11) for primaquine and chloroquine, respectively. Both drugs significantly accelerated the apparent inactivation time constant of I(to). Steady-state inactivation of I(to) was not altered by 30 μM primaquine. In contrast, I(to) recovery from inactivation was prolonged with the appearance of an additional long time constant without a change of the short time constant. Exposure to 1mM chloroquine resulted in a right shift of steady-state inactivation, whereas recovery from inactivation was only mildly affected. Both substances exhibited considerable use dependence. In X. laevis oocytes heterologously expressing hKv4.2+hKChIP2b channels the block by the aminoquinolines was voltage dependent. We conclude that primaquine and chloroquine are open-channel blockers of I(to).
Abstract: Mineralocorticoid receptor (MR) activation modulates cardiac L-type Ca(2+) current (I (CaL)) and transient outward K(+) current (I (to)). The exact circumstances of MR activation, however, remain elusive. Here, we investigate the influence of corticosteroids on MR-mediated changes in cellular electrophysiology. In vitro incubation of adult rat ventricular myocytes with the MR agonist aldosterone (100 nM, 24 h) increased I (CaL) density by 34% (n = 16; p < 0.01). This effect was abrogated by co-incubation with the MR antagonist spironolactone (10 muM). To investigate whether an increase in serum aldosterone concentration is sufficient for an increase in I (CaL) in vivo, rats were subjected to low Na(+) diet (LSD, 0.013% Na(+)) for 28 days. This increased serum aldosterone concentration from 0.19 +/- 0.04 nM (n = 6) in control animals (0.3% Na(+), CSD) to 16.1 +/- 2.1 nM (n = 6; p < 0.0001). Strikingly, I (CaL) density was similar in both CSD and LSD rats (-12.9 +/- 0.9 pA pF(-1), n = 18 and -13.7 +/- 1.1 pA pF(-1), n = 16, respectively), as was I (to) density. In vitro, the glucocorticoid corticosterone (1 microM) also increased I (CaL) and this effect was blocked by spironolactone (10 microM). Co-incubation with corticosterone (1 microM, the normal serum concentration) and aldosterone (100 nM, mimicking low Na(+) intake) did not further increase I (CaL) compared to corticosterone alone. Moreover, co-incubation of myocytes with N-acetylcysteine (10 mM) prevented the aldosterone (100 nM) or corticosterone (1 microM)-induced increase in I (CaL). In conclusion, an increase in serum aldosterone concentration in response to LSD is not sufficient for an increase in I (CaL) density in cardiomyocytes in vivo. This is supported in vitro by the absence of an effect of aldosterone on I (CaL) in the presence of a physiological concentration of corticosterone. Moreover, the cellular redox state may modulate MR activation.
Abstract: BNP (brain-type natriuretic peptide) is a cardiac hormone with systemic haemodynamic effects as well as local cytoprotective and antiproliferative properties. It is induced under a variety of pathophysiological conditions, including decompensated heart failure and myocardial infarction. Since regional hypoxia is a potential common denominator of increased wall stretch and myocardial hypoperfusion, we investigated the direct effects of hypoxia on BNP expression, and the role of the HIF (hypoxia-inducible transcription factor) in BNP regulation. Using an RNase protection assay we found a strong hypoxic induction of BNP mRNA expression in different cell lines and in cultured adult rat cardiomyocytes. Systemic hypoxia and exposure to 0.1% CO induced BNP expression in the rodent myocardium in vivo, although this was at a lower amplitude. BNP promoter-driven luciferase expression increased 10-fold after hypoxic stimulation in transient transfections. Inactivation of four putative HREs (hypoxia-response elements) in the promoter by site-directed mutagenesis revealed that the HRE at -466 nt was responsible for hypoxic promoter activation. A functional CACAG motif was identified upstream of this HRE. The HIF-1 complex bound specifically and inducibly only to the HRE at -466 nt, as shown by EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation). siRNA (small interfering RNA)-mediated knockdown of HIF-1alpha, but not HIF-2alpha, interfered with hypoxic BNP mRNA induction and BNP promoter activation, confirming that BNP is a specific HIF-1alpha target gene. In conclusion, BNP appears to be part of the protective program steered by HIF-1 in response to oxygen deprivation. Induction of BNP may therefore contribute to the potential benefits of pharmacological HIF inducers in the treatment of ischaemic heart disease and heart failure.
Abstract: OBJECTIVE: A reduction of the Ca(2+)-independent transient outward potassium current (I(to)) in epicardial but not in endocardial myocytes of the left ventricle has been observed in cardiac hypertrophy and is thought to contribute to the electrical vulnerability associated with this pathology. METHODS: In the present study we investigated the molecular mechanisms underlying regional alterations in I(to) in hypertrophied hearts of spontaneously hypertensive rats (SHR) using the whole-cell patch-clamp technique, quantitative RT-PCR and heterologous expression of underlying ion channel subunits. RESULTS: I(to) was significantly smaller in epicardial myocytes of SHR than in Wistar-Kyoto (WKY) controls (11.1+/-0.9 pA/pF, n=20 vs. 16.8+/-1.7 pA/pF, n=20, p<0.01), but not different in endocardial myocytes from both groups. Quantitative RT-PCR analysis of the genes encoding I(to) revealed significantly lower levels of Kv4.2 and Kv4.3 mRNA in the epicardial region of SHR rats compared to WKY rats. In contrast, mRNA expression levels of all three splice variants of the beta-subunit KChIP2 were significantly higher in both endo- and epicardial myocytes from SHR than from WKY rats. In parallel, inactivation of I(to), which is negatively modulated by KChIP2, was slowed down in SHR while recovery from inactivation remained unchanged. Heterologous co-expression of increasing amounts of KChIP2b together with a fixed amount of Kv4.2 in Xenopus laevis oocytes revealed a hyperbolic relation of recovery from inactivation and inactivation time constant, demonstrating that KChIP2 preferentially affects inactivation, if its expression level is high. CONCLUSION: These results suggest that downregulation of I(to) in the left ventricle of SHR is mediated by a reduced expression of Kv4.2 and Kv4.3 (but not of KChIP2), whereas the slower inactivation of I(to) can be explained by increased expression levels of KChIP2 in SHR.
Abstract: Inhibition of endothelin-A (ET(A)) receptors has been shown to reduce ventricular electrical abnormalities associated with cardiac failure. In this study, we investigate the effect of ET(A)-receptor inhibition on the development of regional alterations of the transient outward K(+) current (I (to)) in the setting of pressure-induced left ventricular (LV) hypertrophy. Cardiac hypertrophy was induced in female Sprague-Dawley rats by stenosis of the ascending aorta (AS) for 7 days. Treatment with the selective ET(A)-receptor antagonist darusentan (LU135252, 35 mg [kg body weight](-1) day(-1)) was started 1 day before the surgery. AS induced a 46% increase in the relative LV weight (p < 0.001) and caused a significant reduction in I (to) (at +40 mV) in epicardial myocytes (19.5 +/- 1.2 pA pF(-1), n = 32 vs 23.2 +/- 1.2 pA pF(-1), n = 35, p < 0.05). Darusentan further reduced I (to) in AS (15.4 +/- 1.3 pA pF(-1), n = 37, p < 0.05) and sham-operated animals (19.8 +/- 1.6 pA pF(-1), n = 48, ns.). The effects of AS and darusentan on I (to) were significant and independent as tested by two-way analysis of variance. I (to) was not affected in endocardial myocytes. These results indicate that endothelin-1 may exert a tonic effect on the magnitude of I (to) in the epicardial region of the left ventricle but that ET(A)-receptor activation is not necessary for the development of electrical alterations associated with pressure-induced hypertrophy.
Abstract: BACKGROUND/AIMS: Heteromeric KCNEx/KCNQ1 (=KvLQT1, Kv7.1) K(+) channels are important for repolarization of cardiac myocytes, endolymph secretion in the inner ear, gastric acid secretion, and transport across epithelia. They are modulated by pH in a complex way: homomeric KCNQ1 is inhibited by external acidification (low pH(e)); KCNE2/KCNQ1 is activated; and for KCNE1/KCNQ1, variable effects have been reported. Methods: The role of KCNE subunits for the effect of pH(e) on KCNQ1 was analyzed in transfected COS cells and cardiac myocytes by the patch-clamp technique. RESULTS: In outside-out patches of transfected cells, hKCNE2/hKCNQ1 current was increased by acidification down to pH 4.5. Chimeras with the acid-insensitive hKCNE3 revealed that the extracellular N-terminus and at least part of the transmembrane domain of hKCNE2 are needed for activation by low pH(e). hKCNE1/hKCNQ1 heteromeric channels exhibited marked changes of biophysical properties at low pH(e): The slowly activating hKCNE1/hKCNQ1 channels were converted into constitutively open, non-deactivating channels. Experiments on guinea pig and mouse cardiac myocytes pointed to an important role of KCNQ1 during acidosis implicating a significant contribution to cardiac repolarization under acidic conditions. CONCLUSION: External pH can modify current amplitude and biophysical properties of KCNQ1. KCNE subunits work as molecular switches by modulating the pH sensitivity of human KCNQ1.
Abstract: A decrease of the transient outward potassium current (Ito) has been observed in cardiac hypertrophy and contributes to the altered shape of the action potential (AP) of hypertrophied ventricular myocytes. Since the shape and duration of the ventricular AP are important determinants of the Ca2+ influx during the AP (QCa), we investigated the effect of ascending aortic stenosis (AS) on QCa in endo- and epicardial myocytes of the left ventricular free wall using the AP voltage-clamp technique. In sham-operated animals, QCa was significantly larger in endocardial compared to epicardial myocytes (803 +/- 65 fC pF(-1), n = 27 vs. 167 +/- 32 fC pF(-1), n = 38, P < 0.001). Ascending aortic stenosis significantly increased QCa in epicardial myocytes (368 +/- 54 fC pF(-1), n = 42, P < 0.05), but did not alter QCa in endocardial myocytes (696 +/- 65 fC pF(-1), n = 26). Peak and current-voltage relation of the AP-induced Ca2+ current were unaffected by AS. However, the time course of the current-voltage relation was significantly prolonged in epicardial myocytes of AS animals. Model calculations revealed that the increase in QCa can be ascribed to a prolonged opening of the activation gate, whereas an increase in inactivation prevents an excessive increase in QCa. In conclusion, AS significantly increased AP-induced Ca2+ influx in epicardial but not in endocardial myocytes of the rat left ventricle.
