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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-damp 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. [ABSTRACT FROM AUTHOR]

Regulation of the epithelial sodium channel (ENaC) is important for the long-term control of arterial blood pressure as evidenced by gain of function mutations of ENaC causing Liddle’s syndrome, a rare form of hereditary arterial hypertension. In Xenopus laevis oocytes expressing ENaC a spontaneous decline of ENaC currents over time, so-called rundown, is commonly observed. Mechanisms involved in rundown may be physiologically relevant and may be related to feedback regulation of ENaC by intra- or extracellular Na+. We tested the effect of extracellular Na+ removal on ENaC rundown. Spontaneous rundown of ENaC was largely prevented by extracellular Na+ removal and was partially prevented by primaquine suggesting that it is due to endocytic channel retrieval. Liddle’s syndrome mutation caused a reduced rate of rundown, and in oocytes expressing the mutated channel extracellular Na+ removal not only prevented rundown but even increased the ENaC currents (runup). Acute exposure to high extracellular Na+ drastically reduced whole-cell currents and surface expression of wild-type ENaC, while these effects were much smaller in ENaC with Liddle’s syndrome mutation consistent with a stabilization of the mutated channel in the plasma membrane. Interestingly, the apparent intracellular Na+ concentration [Na+]i-app was high (>60 mM) in ENaC-expressing oocytes but rundown was not associated with a further increase in [Na+]i-app. We conclude that the inhibitory effect of extracellular Na+ removal on rundown is due to an inhibition of endocytic ENaC retrieval. [ABSTRACT FROM AUTHOR]

Objective: Several members of the PKD gene family (PKD2, PKDL and PKD2L2) are expressed in the heart. Polycystin-2 and its homologues, which are encoded by these genes, have recently been shown to form Ca2+-regulated nonselective cation channels in heterologous expression systems. Previously, large conductance nonselective cation channels (LCC) have been described in cardiomyocytes, however, their molecular identity remained obscure. We therefore examined whether LCCs may be formed by polycystins. Methods: Myocytes isolated from the rat left ventricle were investigated by the whole-cell patch-clamp technique and single-cell RT-PCR. Results: Application of 10 mM caffeine to the bath solution to increase the intracellular Ca2+ concentration led to activation of LCC in 56% of the myocytes investigated (total n=651), in ∼10%, more than three LCCs were detected. The single channel conductance was ∼300 pS for monovalent cations and the channel was relatively nonselective for the monovalent cations Na+, K+, Li+, and Cs+ and also permeable for the divalent cations Ca2+ and Ba2+, but impermeable for NMDG+ and Cl−. Amiloride (IC50=131±1.1 μM) and millimolar concentrations of the trivalent cations Gd3+ and La3+ inhibited the LCC. Single-cell RT-PCR analysis revealed that mRNA of PKD2 and PKD2L2, but not PKDL or PKD1 are expressed in individual rat left ventricular myocytes. Conclusion: The characteristics of LCC shown in the present study are nearly identical to those observed for polycystin-2 and its homologues suggesting that polycystin-2 or polycystin-2L2 underlie LCC in ventricular myocytes. [Copyright &y& Elsevier]

Introduction: A basic prerequisite for a good surgical outcome in heart surgery is optimal myocardial protection. However, cardioplegia strategies used in adult cardiac surgery are not directly transferable to infant hearts. Paediatric microplegia, analogous to Calafiore cardioplegia used in adult cardiac surgery, offers the advantage of safe myocardial protection without haemodilution. The use of concentration-dependent paediatric microplegia is new in clinical implementation. Material and methods: Paediatric microplegia has been in clinical use in our institution since late 2014. It is applied via an 1/8 inch tube of a S5-HLM roller pump (LivaNova, Italy). As cardioplegic additive, a mixture of potassium (K) 20 mL (2 mmol/mL potassium chloride 14.9% Braun) and magnesium (Mg) 10 mL (4 mmol/mL Mg-sulphate Verla® i. v. 50%) is fixed into a syringe-pump (B. Braun, Germany). This additive is mixed with arterial patient blood from the oxygenator in different flow dependent ratios to form an effective cardioplegia. Technique: After microplegia application of initially 25 mmol/L K with 11 mmol/L Mg for 2 min, a safe cardioplegic cardiac arrest is achieved, which after release of the coronary circulation, immediately returns to a spontaneous cardiac-rhythm. In the case of prolonged aortic clamping, microplegia is repeated every 20 min with a reduction of the application dose of K by 20% and Mg by 30% (20 mmol/L K; 8.5 mmol/L Mg) and a further reduction down to a maintenance dose (15 mmol/L K; 6 mmol/L Mg) after additional 20 min. Summary: The microplegia adapted to the needs of paediatric myocardium is convincing due to its simple technical implementation for the perfusionist while avoiding haemodilution. However, the required intraoperative interval of microplegia of approx. 20 min demands adapted intraoperative management from the surgeon. [ABSTRACT FROM AUTHOR]

In terms of preserving multicellularity and myocardial function in vitro, the cultivation of beating myocardial slices is an emerging technique in basic and translational cardiac research. It can be used, for example, for drug screening or to study pathomechanisms. Here, we describe staining for viable cardiomyocytes based on the immunofluorescence of ryanodine receptors (RyRs) in human and rabbit myocardial slices. Biomimetic chambers were used for culture and measurements of contractile force. Fixable fluorophore-conjugated dextran, entering cells with a permeable membrane, was used for death staining. RyRs, nuclei and the extracellular matrix, including the t-system, were additionally stained and analyzed by confocal microscopy and image processing. We found the mutual exclusion of the RyR and dextran signals in cultivated slices. T-System density and nucleus size were reduced in RyR-negative/dextran-positive myocytes. The fraction of RyR-positive myocytes and pixels correlated with the contractile force. In RyR-positive/dextran-positive myocytes, we found irregular RyR clusters and SERCA distribution patterns, confirmed by an altered power spectrum. We conclude that RyR immunofluorescence indicates viable cardiomyocytes in vibratome-cut myocardial slices, facilitating the detection and differential structural analysis of living vs. dead or dying myocytes. We suggest the loss of sarcoplasmic reticulum integrity as an early event during cardiomyocyte death. [ABSTRACT FROM AUTHOR]

The L-type calcium current (ICaL) is the first step in cardiac excitation–contraction-coupling and plays an important role in regulating contractility, but also in electrical and mechanical remodeling. Primary culture of cardiomyocytes, a widely used tool in cardiac ion channel research, is associated with substantial morphological, functional and electrical changes some of which may be prevented by electrical pacing. We therefore investigated ICaL directly after cell isolation and after 24 h of primary culture with and without regular pacing at 1 and 3 Hz in rat left ventricular myocytes. Moreover, we analyzed total mRNA expression of the pore forming subunit of the L-type Ca2+ channel (cacna1c) as well as the expression of splice variants of its exon 1 that contribute to specificity of ICaL in different tissue such as cardiac myocytes or smooth muscle. 24 h incubation without pacing decreased ICaL density by ~ 10% only. Consistent with this decrease we observed a decrease in the expression of total cacna1c and of exon 1a, the dominant variant of cardiomyocytes, while expression of exon 1b and 1c increased. Pacing for 24 h at 1 and 3 Hz led to a substantial decrease in ICaL density by 30%, mildly slowed ICaL inactivation and shifted steady-state inactivation to more negative potentials. Total cacna1c mRNA expression was substantially decreased by pacing, as was the expression of exon 1b and 1c. Taken together, electrical silence introduces fewer alterations in ICaL density and cacna1c mRNA expression than pacing for 24 h and should therefore be the preferred approach for primary culture of cardiomyocytes. [ABSTRACT FROM AUTHOR]

Mineralocorticoid receptor (MR) activation modulates cardiac L-type Ca2+ 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 μM). 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 μM) also increased I CaL and this effect was blocked by spironolactone (10 μM). Co-incubation with corticosterone (1 μM, 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 μM)-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 FROM AUTHOR]

Abstract: Biophysical and genetic evidence suggests that Kv4–KChIP2 heteromeric ion channels are the molecular correlate of the dominating fast component of the cardiac transient outward K+ current (I tof). Since I tof is one of the essential currents contributing to the shape of the cardiac action potential in the rat, ferret, dog, and human heart, an important implication of this concept is that all ventricular cardiac myocytes from these species express members of the Kv4 and the KChIP2 gene family. To test this prediction, we developed a modified protocol for the collection and subsequent multiplex single-cell RT-PCR amplification of RNA from individual cardiomyocytes isolated from defined regions of the rat left ventricle. All cardiomyocytes investigated (n =61) were positive for α-MHC and Kv4.2 expression. KChIP2 mRNA could also be detected in the vast majority (~87%) of cardiomyocytes. Nearly all of these myocytes co-expressed all three cardiac splice variants of KChIP2. In a small but distinct fraction of myocytes (13%), however, we failed to detect KChIP2 mRNA. The detection thresholds were similar for all target genes and approached a sensitivity of a few molecules of RNA per cell. Our findings support the hypothesis that I tof is mediated by Kv4–KChIP2 heteromeric ion channels in the great majority of cardiac myocytes. A small subpopulation of cardiomyocytes, however, appears to express KChIP2 mRNA at insignificant levels and may therefore use other accessory subunits to generate I tof. [Copyright &y& Elsevier]

The possible involvement of potassium channels in central chemosensitivity, with special reference to the Kir1.1 potassium channel, was investigated by studying the CO2 response of presympathetic neurons in the rostroventrolateral medulla (RVLM) in the absence or presence of various K+ channel inhibitors. Synaptic input to RVLM neurons was blocked by local injection of ω-agatoxin and ω-conotoxin. Activity of RVLM neurons was measured by recording the electrical activity in preganglionic (WR-T3) or postganglionic (renal) sympathetic nerves after perfusion of the lower brainstem via the left vertebral artery with CO2-enriched saline solution. Unspecific K+ channel blockade by BaCl2 reduced the excitatory response of sympathetic activity after CO2-perfusion to 56% of control. A quantitatively similar inhibition of the central CO2 response was obtained after administration of 9-fluorenylmethylchloroformate (FMOC-Cl) which eliminates pH sensitivity of Kir1 and Kir4.1. Furthermore, two structurally different Kir1 inhibiting toxins, tertiapin and Lq2, also reduced the central CO2 response to ∼50% of control. In contrast, charybdotoxin (CTX) had no effect on the CO2 response. Using RT-PCR the expression of mRNA homologous to rat Kir1 mRNA was identified in the cat medulla oblongata. These data suggest that a modulation of potassium channel activity possibly via Kir1 may contribute to central chemosensitivity. [Copyright &y& Elsevier]

Cryopreservation of whole organs and specific tissues is an important and continually expanding field of medicine. The protocols currently used for organ preservation do not ensure survivability and functionality; the protocols for ovarian tissue lead to acceptable outcomes, but these are still capable of further improvement. In general, cryopreservation protocols need to be optimized. One important approach to improving cryopreservation protocols in general involves reducing exposure to cytotoxic cryoprotective agents prior to freezing. This study, therefore, evaluated the real-time tissue penetration of dimethyl sulfoxide, a cryoprotective agent that is widely used in cryopreservation. Dimethyl sulfoxide penetration in rat hearts perfused with a 15% (v/v) dimethyl sulfoxide solution was examined in real-time using dynamic contrast-enhanced micro-computed tomography imaging. Viability of cardiomyocytes was not significantly affected by the dimethyl sulfoxide perfusion procedure. Two different perfusion rates were evaluated and compared with perfusion using a common iodine-based contrast agent (iomeprol). The dynamic contrast-enhanced micro-computed tomography imaging data showed that dimethyl sulfoxide flushes both the extracellular and intracellular spaces in rat heart tissue to 95% equilibration after ≈ 35 s via perfusion. Subsequent wash-out via perfusion is completed to 95% within ≈ 49 s. The equilibration duration routinely used in dimethyl sulfoxide–based protocols for cryopreservation should therefore be questioned. Shorter incubation duration would perhaps be sufficient, as well as being beneficial in relation to cell survivability. It would be helpful to have techniques for non-invasive real-time monitoring of the penetration of cryoprotective agents and such techniques should be used to revise cryopreservation protocols. Switching to perfusion-based equilibration procedures might be beneficial, if feasible. [ABSTRACT FROM AUTHOR]

Regional differences in ion channel activity in the heart control the sequence of repolarization and may contribute to differences in contraction. Corticosteroids such as aldosterone or corticosterone increase the L-type Ca2+ current (ICaL) in the heart via the mineralocorticoid receptor (MR). Here, we investigate the differential impact of corticosteroid-mediated increase in ICaL on action potentials (AP), ion currents, intracellular Ca2+ handling and contractility in endo- and epicardial myocytes of the rat left ventricle. Dexamethasone led to a similar increase in ICaL in endocardial and epicardial myocytes, while the K+ currents Ito and IK were unaffected. However, AP duration (APD) and AP-induced Ca2+ influx (QCa) significantly increased exclusively in epicardial myocytes, thus abrogating the normal differences between the groups. Dexamethasone increased Ca2+ transients, contractility and SERCA activity in both regions, the latter possibly due to a decrease in total phospholamban (PLB) and an increase PLBpThr17. These results suggest that corticosteroids are powerful modulators of ICaL, Ca2+ transients and contractility in both endo- and epicardial myocytes, while APD and QCa are increased in epicardial myocytes only. This indicates that increased ICaL and SERCA activity rather than QCa are the primary drivers of contractility by adrenocorticoids. [ABSTRACT FROM AUTHOR]

KCNQ1 (Kv7.1) proteins form a homotetrameric channel, which produces a voltage-dependent K + current. Co-assembly of KCNQ1 with the auxiliary β-subunit KCNE1 strongly up-regulates this current. In cardiac myocytes, KCNQ1/E1 complexes are thought to give rise to the delayed rectifier current I Ks , which contributes to cardiac action potential repolarization. We report here that the type I membrane protein BACE1 (β-site APP-cleaving enzyme 1), which is best known for its detrimental role in Alzheimer's disease, but is also, as reported here, present in cardiac myocytes, serves as a novel interaction partner of KCNQ1. Using HEK293T cells as heterologous expression system to study the electrophysiological effects of BACE1 and KCNE1 on KCNQ1 in different combinations, our main findings were the following: (1) BACE1 slowed the inactivation of KCNQ1 current producing an increased initial response to depolarizing voltage steps. (2) Activation kinetics of KCNQ1/E1 currents were significantly slowed in the presence of co-expressed BACE1. (3) BACE1 impaired reconstituted cardiac I Ks when cardiac action potentials were used as voltage commands, but interestingly augmented the I Ks of ATP-deprived cells, suggesting that the effect of BACE1 depends on the metabolic state of the cell. (4) The electrophysiological effects of BACE1 on KCNQ1 reported here were independent of its enzymatic activity, as they were preserved when the proteolytically inactive variant BACE1 D289N was co-transfected in lieu of BACE1 or when BACE1-expressing cells were treated with the BACE1-inhibiting compound C3. (5) Co-immunoprecipitation and fluorescence recovery after photobleaching (FRAP) supported our hypothesis that BACE1 modifies the biophysical properties of I Ks by physically interacting with KCNQ1 in a β-subunit-like fashion. Strongly underscoring the functional significance of this interaction, we detected BACE1 in human iPSC-derived cardiomyocytes and murine cardiac tissue and observed decreased I Ks in atrial cardiomyocytes of BACE1-deficient mice. [ABSTRACT FROM AUTHOR]

Lipid rafts are cholesterol-enriched microdomains of the cell membrane. Here we investigate the localization of the pore forming K-channel α-subunit Kv4.2 and the β-subunit KChIP2, underlying the transient outward K current ( I), in lipid rafts in left ventricular myocytes. Furthermore, we explored the impact of membrane cholesterol depletion (using 20 mM methyl-beta-cyclodextrin (MBCD)) on K outward currents. Cholesterol-saturated MBCD (20 mM) served as control. Myocytes were isolated from the left ventricular free wall of Wistar rats. The Triton X-100 (4 °C) insoluble fraction of whole cell protein was analyzed by sucrose density gradient centrifugation followed by Western blot. Kv4.2 and KChIP2 were partially detected in low-density fractions (lipid rafts). MBCD treatment (5 min) resulted in a shift of Kv4.2 and KChIP2 towards high-density fractions. K currents were assessed by whole-cell patch-clamp. MBCD treatment resulted in a 29 ± 3 % decrease in I (20.0 ± 1.6pApF vs. 28.5 ± 2.0pApF, n = 15, p < 0.001, V = 40 mV) within 5 min. Control solution resulted in a significantly smaller reduction in I (17 ± 3 %, p < 0.001, p < 0.01 compared with MBCD). MBCD induced a 38 ± 9 % increase in the non-inactivating current component ( I) (10.1 ± 0.6pApF vs. 7.6 ± 0.4pApF, n = 15, p < 0.001). This effect was absent in control solution. The increase in I was not sensitive to 100 μM 4-aminopyridine or 20 mM tetraethylammonium, making a contribution of Kv1.5 or Kv2.1 unlikely. In conclusion, in rat ventricular cardiomyocytes, a fraction of Kv4.2 and KChIP2 is localized in lipid rafts. Membrane cholesterol depletion results in ~12 % net reduction of I, a redistribution of the channel proteins Kv4.2 and KChIP2 and an increased delayed rectifier current. [ABSTRACT FROM AUTHOR]

Pressure overload is associated with cardiac hypertrophy and electrical remodeling. Here, we investigate the effects of the antioxidant N-acetylcysteine (NAC) on the cellular cardiac electrophysiology of female Sprague-Dawley rats with ascending aortic stenosis (AS). Rats were treated with NAC (1 g/kg body weight) or control solution 1 week before the intervention and in the week following AS or sham operation. Seven days after the operation, blood pressure and left ventricular pressure were measured before the heart was excised. Single cells were isolated from epicardial and endocardial layers of the left ventricular free wall and investigated using the whole-cell patch-clamp technique. Systolic blood pressure and left ventricular peak pressure were not significantly altered in the NAC group. NAC reduced the increase ( p < 0.001) in the relative left ventricular weight ( p < 0.05) as well as the increase ( p < 0.001) in cell capacitance in epicardial ( p < 0.05), but not in endocardial myocytes of AS animals. The L-type Ca current ( I) was significantly increased by AS in epicardial (+19 % at 0 mV, p < 0.01) but not in endocardial myocytes. NAC completely prevented this increase in I ( p < 0.01). The current density of the transient outward K current ( I) was not affected by AS or NAC. Action potential duration to 90 % repolarization was significantly prolonged in epicardial ( p < 0.01) as well as in endocardial ( p < 0.001) cells of AS animals. NAC prevented the AP prolongation in epicardial myocytes only ( p < 0.05). We conclude that reducing oxidative stress in pressure overload can prevent electrical remodeling and ameliorate hypertrophy in epicardial but not in endocardial myocytes. [ABSTRACT FROM AUTHOR]

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 (Ito) 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 Ito. At +60mV, primaquine blocked Ito amplitude (defined as the current inactivating during a test pulse of 600ms duration) with an IC50 of 118±8μM. Ito charge was blocked with an IC50 of 33±2μM (n=42), indicating open channel block. Chloroquine blocked Ito amplitude with an IC50 of 4.6±0.9mM, while the IC50 for Ito charge was 439±63μM (n=23). The kinetic analysis of the onset of block revealed Kd values of 52±8μM (n=18) and 520±60μM (n=11) for primaquine and chloroquine, respectively. Both drugs significantly accelerated the apparent inactivation time constant of Ito. Steady-state inactivation of Ito was not altered by 30μM primaquine. In contrast, Ito 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 Ito. [ABSTRACT FROM AUTHOR]

The α1-adrenoceptor as well as the AT1- and the ETA-receptor couple to G-proteins of the Gαq/11 family and contribute to the regulation of the transient outward K+ current (Ito,f) under pathological conditions such as cardiac hypertrophy or failure. This suggests an important role of Gαq/11-signalling in the physiological regulation of Ito,f. Here, we investigate mice deficient of the Gα11 protein (gna11−/−) to clarify the physiological role of Gα11 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 Ito,f than controls (52.6 ± 4.1 pApF−1, n = 20 vs 42.7 ± 2.8 pApF−1, n = 26, p < 0.05). Endocardial Ito,f was similar in gna11−/− mice and controls. With the except of minor changes in endocardial myocytes, Ito,f kinetics were similar in both groups. In the epicardial layer, western blot analysis revealed a 19% higher expression of the K+-channel α-subunit Kv4.2 in gna11−/− mice than in wild type (wt; p < 0.05). The β-subunit KChIP2b was upregulated by 102% in epicardial myocytes of gna11−/− mice ( p < 0.01, n = 4). Consistent with the difference in Ito,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 Gα11-coupled signalling is a central pathway in the regulation of Ito,f. It physiologically exerts a tonic inhibitory influence on the expression of Ito,f and thereby contributes to the regulation of cardiac repolarisation. [ABSTRACT FROM AUTHOR]

Abstract: Cardiac unloading with left ventricular assist devices is increasingly used to treat patients with severe heart failure. Unloading has been shown to improve systolic and diastolic function, but its impact on the repolarization of left ventricular myocytes is not known. Unloaded hearts exhibit similar patterns of gene expression as hearts subjected to an increased hemodynamic load. We therefore hypothesized that cardiac unloading also replicates the alterations in action potential and underlying repolarizing ionic currents found in pressure-overload induced cardiac hypertrophy. Left ventricular unloading was induced by heterotopic heart transplantation in syngenic male Lewis rats. Action potentials and underlying K+ and Ca2+ currents were investigated using whole-cell patch-clamp technique. Real-time RT-PCR was used to quantify mRNA expression of Kv4.2, Kv4.3, and KChIP2. Unloading markedly prolonged cardiac action potentials and suppressed the amplitude of several repolarizing K+ currents, in particular of the transient outward K+ current I to, in both, epicardial and endocardial myocytes. The reduction of I to was associated with significantly lower levels of Kv4.2 and Kv4.3 mRNAs in epicardial myocytes, and of KChIP2 mRNA in endocardial myocytes. Concomitantly, the L-type Ca2+ current was increased in myocytes of unloaded hearts. Collectively, these results show that left ventricular unloading induces a profound remodelling of cardiac repolarization with action potential prolongation, downregulation of repolarizing K+ currents and upregulation of the L-type Ca2+ current. This indicates that unloaded rat hearts in vivo express a hypertrophic phenotype of cardiac repolarization at the cellular and the molecular level. [Copyright &y& Elsevier]

Inhibition of endothelin-A (ETA) receptors has been shown to reduce ventricular electrical abnormalities associated with cardiac failure. In this study, we investigate the effect of ETA-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 ETA-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 ETA-receptor activation is not necessary for the development of electrical alterations associated with pressure-induced hypertrophy. [ABSTRACT FROM AUTHOR]

Abstract: Objective: A reduction of the Ca2+-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 β-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. [Copyright &y& Elsevier]

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 pHe); KCNE2/KCNQ1 is activated; and for KCNE1/KCNQ1, variable effects have been reported. Methods: The role of KCNE subunits for the effect of pHe 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 pHe. hKCNE1/hKCNQ1 heteromeric channels exhibited marked changes of biophysical properties at low pHe: 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. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]