Murine Ventricular Myocytes Research Articles

Modulation of cardiac mechanosensitive ion channels involves superoxide, nitric oxide and peroxynitrite

In murine ventricular myocytes, activation of mechanosensitive ion channels (MSCs) includes activation of non-selective cation currents and deactivation of inwardly rectifying potassium currents. Using pharmacological inhibitors and knockout models, we analyzed signaling steps that are critical to transduce the mechanical signal (stretch) into electrophysiological events (MSC). We provide evidence for an activation of NAD(P)H oxidase and NOS3 in response to stretch putatively via the angiotensin II receptor type 1. The involvement of superoxide and nitric oxide was verified by the block of MSC using specific scavengers (tiron and PTIO, respectively). Superoxide and nitric oxide are known to combine very rapidly to form peroxynitrite. Accordingly, MSC were blocked by the peroxynitrite scavenger uric acid and could be mimicked by application of exogenous peroxynitrite. Peroxynitrite formation may activate phospholipases generating amphipaths that modulates channel function via changing the curvature of the surrounding lipid bilayer. This conclusion is supported by our findings that MSC were suppressed by inhibitors of phospholipases but could be mimicked by exogenous phospholipases or by amphipaths (oleic acid, Triton X-100).

Mechanical deformation of ventricular myocytes modulates both TRPC6 and Kir2.3 channels

Cardiomyocytes respond to mechanical stretch with an increase [Ca2+]i. Here, we analyzed which ion channels could mediate this effect. Murine ventricular myocytes were attached to a glass coverslip and a cell-attached glass stylus sheared the upper cell part versus the attached cell bottom. At negative clamp potentials, stretch induced inward currents that increased with the extent of stretch and reversed within 2 min after relaxation from stretch. Stretch activated a nearly voltage-independent GsMTx-4-sensitive non-selective cation conductance Gns, antibodies against TRPC6 prevented Gns activation. In addition, stretch deactivated a Cs+-sensitive inwardly rectifying potassium conductance GK1, antibodies against Kir2.3 inhibited this effect. Immunolabeling localized TRPC6 and Kir2.3 in T-tubular membranes, and stretch-induced changes in membrane currents were absent in cells whose T-tubules had been removed. In absence of stretch, we could activate Gns and deactivate GK1 by 1-oleoyl-2-acetyl-sn-glycerol (OAG) and other amphipaths. We interpret that the function of TRPC6 and Kir2.3 channels is controlled by both tension and curvature of the surrounding lipid bilayer that are changed by incorporation of amphipaths. Stretch-activation of TRPC6 channels may increase Ca2+ influx directly and indirectly, by membrane depolarization (activation of voltage-gated Ca2+ channels) and by elevated [Na+]i (augmented Na+,Ca2+-exchange).

Inhibitory phosphorylation of TASK‐1 is associated with atrial fibrillation

Activation of the platelet‐activating factor (PAF) receptor leads to inhibition of TASK‐1, a two pore‐domain potassium channel. PAF‐induced TASK‐1 inhibition requires the activity of PKCε and causes repolarization abnormalities in isolated murine ventricular myocytes. We have previously shown that TASK‐1 current is absent from atrial myocytes derived from humans suffering from atrial fibrillation (AF). We now show that AF‐associated loss of TASK‐1 current occurs in spite of significantly higher levels of channel protein expression in AF atrium as compared to atrial tissue from patients in normal sinus rhythm, suggesting regulation by post‐translational modification. TASK‐1 current was restored in atrial myocytes by PP2A treatment, showing that inhibition in AF was phosphorylation‐dependent. We have made a construct of human TASK‐1 carboxy‐terminus fused to glutathione‐S‐transferase and made site‐directed mutants of potential PKC sites. Kinase reactions of these constructs with PKCε identified the phosphorylation site as T383, a result that was confirmed by LC‐MS/MS analysis. Similar mutations in the full length channel abrogated the ability of PAF to inhibit TASK‐1 current in whole cell patch clamp studies. These data provide an exciting new marker, and possible therapeutic target, for AF, the most common human arrhythmia.Supported by NIH grants 5R01HL070105‐04, 5TL1RR024158‐02 and Servier

Characterization of the cardiac sodium channel SCN5A mutation, N1325S, in single murine ventricular myocytes

The N1325S mutation in the cardiac sodium channel gene SCN5A causes the type-3 long-QT syndrome but the arrhythmogenic trigger associated with N1325S has not been characterized. In this study, we investigated the triggers for cardiac events in the expanded N1325S family. Among 11 symptomatic patients with document triggers, six died suddenly during sleep or while sitting (bradycardia-induced trigger), three died suddenly, and two developed syncope due to stress and excitement (non-bradycardia-induced). Patch-clamping studies revealed that the late sodium current (INa,L) generated by mutation N1325S in ventricular myocytes from TG-NS/LQT3 mice was reduced with increased pacing, which explains bradycardia-induced mortalities in the family. The non-bradycardic triggers are related to the finding that APD became prolonged and unstable at increasing rates, often with alternating repolarization phases which was corrected with verapamil. This implies that Ca2+ influx and intracellular Ca2+ ([Ca2+]i) ions are involved and that [Ca2+]i inhomogeneity may be the underlying mechanisms behind non-bradycardia LQT3 arrhythmogenesis associated with mutation N1325S.

Intermedin (adrenomedullin-2) enhances cardiac contractile function via a protein kinase C- and protein kinase A-dependent pathway in murine ventricular myocytes

Intermedin (IMD), also called adrenomedullin-2, is a 47-amino acid peptide from the calcitonin gene-related peptide (CGRP)/adrenomedullin family of peptides. Recent studies suggest that IMD may participate in the regulation of cardiovascular function and fluid and electrolyte homeostasis. To evaluate the role of IMD on cardiomyocyte contractile function, electrically paced murine ventricular myocytes were acutely exposed to IMD, and the following indexes were determined: peak shortening (PS), time to PS, time-to-90% relengthening, and maximal velocity of shortening and relengthening. Intracellular Ca(2+) was assessed using fura 2-AM fluorescent microscopy. Our results revealed that IMD (10 pM to 10 nM) significantly increased PS and maximal velocity of shortening and relengthening in ventricular myocytes, the maximal effect of which (approximately 46%) was somewhat comparable to those elicited by CGRP (1 nM) and adrenomedullin (100 nM). Exposure of IMD significantly shortened time-to-90% relengthening without affecting time to PS, similar to CGRP and adrenomedullin. IMD also enhanced intracellular Ca(2+) release, with a maximal increase of approximately 50%, and facilitated the intracellular Ca(2+) decay rate. The IMD-induced effects were abolished by the protein kinase C inhibitor chelerythrine (1 microM), downregulation of protein kinase C using phorbol 12-myristate 13-acetate (1 microM), and the protein kinase A inhibitor H89 (1 microM). Our data suggest that IMD acutely augments cardiomyocyte contractile function through, at least in part, a protein kinase C- and protein kinase A-dependent mechanism.

Dynamic model for ventricular junctional conductance during the cardiac action potential

The ventricular action potential was applied to paired neonatal murine ventricular myocytes in the dual whole cell configuration. During peak action potential voltages >100 mV, junctional conductance (g(j)) declined by 50%. This transjunctional voltage (V(j))-dependent inactivation exhibited two time constants that became progressively faster with increasing V(j). G(j) returned to initial peak values during action potential repolarization and even exceeded peak g(j) values during the final 5% of repolarization. This facilitation of g(j) was observed <30 mV during linearly decreasing V(j) ramps. The same behavior was observed in ensemble averages of individual gap junction channels with unitary conductances of 100 pS or lower. Immunohistochemical fluorescent micrographs and immunoblots detect prominent amounts of connexin (Cx)43 and lesser amounts of Cx40 and Cx45 proteins in cultured ventricular myocytes. The time dependence of the g(j) curves and channel conductances are consistent with the properties of predominantly homomeric Cx43 gap junction channels. A mathematical model depicting two inactivation and two recovery phases accurately predicts the ventricular g(j) curves at different rates of stimulation and repolarization. Functional differences are apparent between ventricular myocytes and Cx43-transfected N2a cell gap junctions that may result from posttranslational modification. These observations suggest that gap junctions may play a role in the development of conduction block and the genesis and propagation of triggered arrhythmias under conditions of slowed conduction (<10 cm/s).

Activation of Protein Kinase C ϵ Inhibits the Two-pore Domain K+ Channel, TASK-1, Inducing Repolarization Abnormalities in Cardiac Ventricular Myocytes

Activation of the platelet-activating factor (PAF) receptor leads to a decrease in outward current in murine ventricular myocytes by inhibiting the TASK-1 channel. TASK-1 carries a background or "leak" current and is a member of the two-pore domain potassium channel family. Its inhibition is sufficient to delay repolarization, causing prolongation of the action potential duration, and in some cases, early after depolarizations. We set out to determine the cellular mechanisms that control regulation of TASK-1 by PAF. Inhibition of TASK-1 via activation of the PAF receptor is protein kinase C (PKC)-dependent. Using isoform-specific PKC inhibitor or activator peptides in patch clamp experiments, we now demonstrate that activation of PKCepsilon is both necessary and sufficient to regulate murine TASK-1 current in a heterologous expression system and to induce repolarization abnormalities in isolated myocytes. Furthermore, site-directed mutagenesis studies have identified threonine 381, in the C-terminal tail of murine TASK-1, as a critical residue in this regulation.

The significance of computational modelling in murine cardiac ventricular cells

Abstract Five decades of histological, electrophysiological, pharmacological and biochemical investigations exist, but relatively little is known regarding the ionic mechanisms underlying the action potential variations in the ventricle associated with healthy and disease conditions. The computational modelling in murine ventricular myocytes can complement our knowledge of the experimental data and provide us with more quantitative descriptions in understanding different conditions related to normal and disease conditions. This paper initially reviews the theoretical modelling for cardiac ventricular action potentials of various species and the related experimental work. It then focuses on the progress of computational modelling of cardiac ventricular cells for normal, diabetic and spontaneously hypertensive rats. Also presented is the recent modelling efforts of the action potential in mouse ventricular cells. The computational insights gained into the ionic mechanisms in rodents will enhance our understanding of the heart and provide us with new knowledge for future studies to treat cardiac diseases in children and adults.

The significance of computational modelling in murine cardiac ventricular cells

Five decades of histological, electrophysiological, pharmacological and biochemical investigations exist, but relatively little is known regarding the ionic mechanisms underlying the action potential variations in the ventricle associated with healthy and disease conditions. The computational modelling in murine ventricular myocytes can complement our knowledge of the experimental data and provide us with more quantitative descriptions in understanding different conditions related to normal and disease conditions. This paper initially reviews the theoretical modelling for cardiac ventricular action potentials of various species and the related experimental work. It then focuses on the progress of computational modelling of cardiac ventricular cells for normal, diabetic and spontaneously hypertensive rats. Also presented is the recent modelling efforts of the action potential in mouse ventricular cells. The computational insights gained into the ionic mechanisms in rodents will enhance our understanding of the heart and provide us with new knowledge for future studies to treat cardiac diseases in children and adults.

Computational Modeling of Cardiac Ventricular Action Potentials in Rat and Mouse: Review

Little is known about the ionic mechanisms underlying the action potential heterogeneity in ventricle-associated healthy and disease conditions, even though five decades of histological, electrophysiological, pharmacological, and biochemical investigations exist. The computational modeling in murine ventricular myocytes can complement our knowledge of the experimental data and provide us with more quantitative descriptions in understanding different conditions related to normal and disease conditions. This paper initially reviews the theoretical modeling for cardiac ventricular action potentials of various species and the related experimental work. It then presents the progress of the computational modeling of cardiac ventricular cells for normal, diabetic, and spontaneously hypertensive rats. The paper also introduces recent modeling efforts for the action potential heterogeneity in mouse ventricular cells. The computational insights gained into the ionic mechanisms in rodents will continue to enhance our understanding of the heart and provide us with new knowledge for future studies to treat cardiac diseases in children and adults. Because the dissemination of computational models is very important, we continue to disseminate these models by iCell, the interactive cell modeling resource. iCell (http://ssd1.bme.memphis.edu/icell/) has been developed as a simulation-based teaching and learning tool for electrophysiology and contains JAVA applets that present models of various cardiac cells and neurons and simulation data of their bioelectric activities at cellular level.

The Significance of Computational Modelling in Murine Cardiac Ventricular Cells

Five decades of histological, electrophysiological, pharmacological and biochemical investigations exist, but relatively little is known regarding the ionic mechanisms underlying the action potential variations in the ventricle associated with healthy and disease conditions. The computational modelling in murine ventricular myocytes can complement our knowledge of the experimental data and provide us with more quantitative descriptions in understanding different conditions related to normal and disease conditions. This paper initially reviews the theoretical modelling for cardiac ventricular action potentials of various species and the related experimental work. It then focuses on the progress of computational modelling of cardiac ventricular cells for normal, diabetic and spontaneously hypertensive rats. Also presented is the recent modelling efforts of the action potential in mouse ventricular cells. The computational insights gained into the ionic mechanisms in rodents will enhance our understanding of the heart and provide us with new knowledge for future studies to treat cardiac diseases in children and adults.

Tyrosine kinases inhibitors block Fas-mediated deleterious effects in normoxic and hypoxic ventricular myocytes

Experimental evidences suggest an important role for Fas receptor activation in a wide range of myocardial pathologies, which are associated with a variety of arrhythmias and mechanical dysfunction. Our recent studies have shown that Fas-mediated arrhythmias and mechanical disturbances of ventricular myocytes can be accounted for by activation of the phospholipase C–1,4,5-inositol triphosphate–intracellular calcium release (PLC–1,4,5–IP 3–[Ca 2+] i) cascade, which is responsible for attenuating the transient outward current ( I to) and augmenting the L-type Ca 2+ current ( I Ca,L). We have also shown that whereas ventricular myocytes are resistant to Fas-mediated apoptosis, hypoxia predisposes myocytes to apoptosis induced by Fas activation by shifting the balance between pro-apoptotic and anti-apoptotic proteins towards the former. Since protein tyrosine phosphorylation has been shown to be involved in Fas signaling, we have hypothesized that inhibiting tyrosine kinases will attenuate Fas-mediated effects in ventricular myocytes in normoxic and hypoxic conditions. Therefore, we tested the effect of the tyrosine kinases inhibitors, genistein (50 μmol/l) and herbimycin A (50 μg/ml), on the abovementioned Fas-mediated effects in cultured neonatal rat ventricular myocytes (NRVM) and in freshly isolated adult murine ventricular myocytes. Fas receptor was activated by incubating NRVM with recombinant Fas ligand (rFasL, 50 ng/ml) and enhancing antibody (1 μg/ml), or by incubation of murine ventricular myocytes with the Fas-activating antibody Jo2 (10 μg/ml). The major findings were that genistein prevented Fas-mediated increase in 1,4,5-IP 3 production in NRVM (quantified by ion-exchange chromatography): 216 ± 41 counts per minute (cpm) in control, 605 ± 184 cpm in FasL-treated cardiomyocytes and 137 ± 51 cpm in rFasL + genistein-treated cultures. Accordingly, genistein or herbimycin A abolished the diastolic [Ca 2+] i-rise (as measured by fura-2 fluorescence) and arrhythmogenic activity in both rat and murine ventricular myocytes, and the Fas-mediated changes in I to and I Ca,L in murine ventricular myocytes. Importantly, genistein attenuated Fas-mediated apoptosis in hypoxic (22 h in 1% O 2) NRVM; the apoptotic ratios as measured by DAPI fluorescence assay were: 4.6 ± 1.0% in control, 12.5 ± 3.0% in rFasL and 7.3 ± 1.6% in rFasL + genistein-treated NRVM. This prevention of apoptosis by tyrosine kinases blockade was accompanied by inhibition of hypoxia-induced increased Fas expression and decreased expression of the anti-apoptotic protein xIAP. In conclusion, our findings indicate that tyrosine phosphorylation is involved in Fas signaling in ventricular myocytes, and can, therefore, serve as a novel potential target for attenuating Fas-mediated dysfunction in normoxic and hypoxic myocardium.

Block of the background K(+) channel TASK-1 contributes to arrhythmogenic effects of platelet-activating factor.

Platelet-activating factor (PAF), an inflammatory phospholipid, induces ventricular arrhythmia via an unknown ionic mechanism. We can now link PAF-mediated cardiac electrophysiological effects to inhibition of a two-pore domain K(+) channel [TWIK-related acid-sensitive K(+) background channel (TASK-1)]. Superfusion of carbamyl-PAF (C-PAF), a stable analog of PAF, over murine ventricular myocytes causes abnormal automaticity, plateau phase arrest of the action potential, and early afterdepolarizations in paced and quiescent cells from wild-type but not PAF receptor knockout mice. C-PAF-dependent currents are insensitive to Cs(+) and are outwardly rectifying with biophysical properties consistent with a K(+)-selective channel. The current is blocked by TASK-1 inhibitors, including protons, Ba(2+), Zn(2+), and methanandamide, a stable analog of the endogenous lipid ligand of cannabinoid receptors. In addition, when TASK-1 is expressed in CHO cells that express an endogenous PAF receptor, superfusion of C-PAF decreases the expressed current. Like C-PAF, methanandamide evoked spontaneous activity in quiescent myocytes. C-PAF- and methanandamide-sensitive currents are blocked by a specific protein kinase C (PKC) inhibitor, implying overlapping signaling pathways. In conclusion, C-PAF blocks TASK-1 or a closely related channel, the effect is PKC dependent, and the inhibition alters the electrical activity of myocytes in ways that would be arrhythmogenic in the intact heart.

Cardiac nitric oxide synthase 1 regulates basal and beta-adrenergic contractility in murine ventricular myocytes.

Evidence indicates that myocardial NO production can modulate contractility, but the source of NO remains uncertain. Here, we investigated the role of a type 1 NO synthase isoform (NOS1), which has been recently localized to the cardiac sarcoplasmic reticulum, in the regulation of basal and beta-adrenergic myocardial contraction. Contraction was assessed in left ventricular myocytes isolated from mice with NOS1 gene disruption (NOS1(-/-) mice) and their littermate controls (NOS1(+/+) mice) at 3 stimulation frequencies (1, 3, and 6 Hz) in basal conditions and during beta-adrenergic stimulation with isoproterenol (2 nmol/L). In addition, we examined the effects of acute specific inhibition of NOS1 with vinyl-L-N-5-(1-imino-3-butenyl)-L-ornithine (L-VNIO, 500 micromol/L). NOS1((-/-)) myocytes exhibited greater contraction at all frequencies (percent cell shortening at 6 Hz, 10.7+/-0.92% in NOS1(-/-) myocytes versus 7.21+/-0.8% in NOS1(+/+) myocytes; P<0.05) with a flat frequency-contraction relationship. Time to 50% relaxation was increased in NOS1(-/-) myocytes at all frequencies (at 6 Hz, 26.53+/-1.4 ms in NOS1(-/-) myocytes versus 21.27+/-1.3 ms in NOS1(+/+) myocytes; P<0.05). L-VNIO prolonged time to 50% relaxation at all frequencies (at 6 Hz, 21.28+/-1.7 ms in NOS1(+/+) myocytes versus 26.45+/-1.4 ms in NOS1(+/+)+L-VNIO myocytes; P<0.05) but did not significantly increase basal contraction. However, both NOS1(-/-) myocytes and NOS1(+/+) myocytes treated with L-VNIO showed a greatly enhanced contraction in response to beta-adrenergic stimulation (percent increase in contraction at 6 Hz, 25.2+/-10.8 in NOS1(+/+) myocytes, 68.2+/-11.2 in NOS1(-/-) myocytes, and 65.1+/-13.2 in NOS1(+/+)+L-VNIO myocytes; P<0.05). NOS1 disruption enhances basal contraction and the inotropic response to beta-adrenergic stimulation in murine ventricular myocytes. These findings indicate that cardiac NOS1-derived NO plays a significant role in the autocrine regulation of myocardial contractility.

Effects of PP1/PP2A inhibitor calyculin A on the E-C coupling cascade in murine ventricular myocytes.

Calyculin A was used to examine the importance of phosphatases in the modulation of cardiac contractile magnitude in the absence of any neural or humoral stimulation. Protein phosphatase (PP)1 and PP2A activity, twitch contractions, intracellular Ca(2+) concentration ([Ca(2+)](i)) transients, action potentials, membrane currents, and myofilament Ca(2+) sensitivity were measured in isolated mouse ventricular myocytes. Calyculin A (125 nM) inhibited PP1 and PP2A by 50% and 85%, respectively, whereas it doubled the twitch magnitude and increased twitch duration by 50% in field-stimulated cells. Calyculin A-evoked increases in L-type Ca(2+) current (70%) and the resulting [Ca(2+)](i) transient (83%) explain the positive inotropic response. However, increases in twitch and action potential durations did not result from increased myofilament Ca(2+) sensitivity or K(+) current inhibition, respectively. Comparison of the effects of calyculin A and isoproterenol on [Ca(2+)](i) transients and twitch contractions revealed that calyculin A had a much smaller lusitropic effect than the beta-agonist, indicating that calyculin A did not significantly increase sarcoplasmic reticulum Ca(2+) reuptake. Thus while cardiac contractile magnitude is controlled by a steady-state kinase/phosphatase balance, this regulation is not equally operative at all of the steps in the excitation-contraction coupling pathway and may in fact be most important to the regulation of the L-type Ca(2+) channel.

Electrophysiologic perturbations and arrhythmogenic activity caused by activation of the Fas receptor in murine ventricular myocytes: role of the inositol trisphosphate pathway.

Experimental evidence suggests a major role for Fas receptor activation in a wide range of myocardial pathologies. Because clinical situations, which are likely to be associated with Fas activation, are accompanied by a variety of ventricular arrhythmias, the major goal of this study was to investigate the ionic mechanisms responsible for these phenomena. To delineate the origin of Fas-mediated electrophysiologic perturbations, the transient outward K+ current I(to) and the L-type Ca2+ current I(Ca,L) were studied in murine ventricular myocytes treated with the Fas-activating monoclonal antibody Jo2. Jo2 decreased I(to) (4.36 +/- 1.2 pA/pF vs 17.48 +/- 2.36 pA/pF in control, V(M) = +50 mV; P < 0.001) and increased I(Ca,L) (-13.17 +/- 1.38 pA/pF vs -3.94 +/- 0.78 pA/pF in control, V(M) = 0 mV; P < 0.001). Pretreatment of ventricular myocytes with ryanodine or thapsigargin prevented the electrophysiologic effects of Jo2, suggesting that [Ca2+]i elevation is important for Fas-mediated action. In agreement with our previous studies demonstrating dependence of Fas-based myocyte dysfunction on an intact inositol trisphosphate (1,4,5-IP3) pathway, the effects of Jo2 on I(to) and I(Ca,L) were prevented by the phospholipase C (generates 1,4,5-IP3) blocker U73122, and by xestospongin C (tested with I(to)), a specific blocker of IP3-operated sarcoplasmic reticulum Ca2+ release channels. Furthermore, intracellular perfusion with 1,4,5-IP3, but not with 1,3,4-IP3, caused electrophysiologic effects resembling those of Jo2. Decreased I(to) and increased I(Ca,L) underlie Fas-induced action potential alterations and arrhythmias in murine ventricular myocytes, effects that appear to be mediated by 1,4,5-IP3-induced intracellular calcium release.

Galpha(i2) but not Galpha(i3) is required for muscarinic inhibition of contractility and calcium currents in adult cardiomyocytes.

Parasympathetic stimulation of the heart acts through M(2)-muscarinic acetylcholine receptors to regulate ion channel activity and subsequent inotropic status. Although muscarinic signal transduction is mediated via pertussis toxin-sensitive G proteins Galpha(i/o), the specific signal transduction requirements of Galpha(i2) and Galpha(i3) in mediating muscarinic regulated L-type calcium currents (I(Ca, L)), intracellular calcium, and cell contractility remain to be determined. Adult ventricular myocytes were isolated from Galpha(i2)-null mice, Galpha(i3)-null mice, and their wild-type littermates. Cell shortening, intracellular calcium levels, and I(Ca, L) were all measured in response to isoproterenol, a beta-adrenergic receptor agonist, and carbachol, a cholinergic receptor agonist. With isoproterenol stimulation, myocytes from all groups demonstrated a marked increase in calcium currents, correlating with augmented intracellular calcium transient amplitude and cell shortening. Carbachol significantly attenuated the isoproterenol response in wild-type and Galpha(i3)-null cells but had no effect in Galpha(i2)-null cells. This study demonstrates that Galpha(i2), but not Galpha(i3), is required for muscarinic inhibition of the beta-adrenergic response in adult murine ventricular myocytes.

Sarcoplasmic Reticulum Function in Murine Ventricular Myocytes Overexpressing SR CaATPase

To examine the effects of the overexpression of sarcoplasmic reticulum (SR) CaATPase on function of the SR and Ca2+homeostasis, we measured [Ca2+]itransients (fluo-3), and L-type Ca2+currents (ICa,L), Na/Ca exchanger currents (INa/Ca), and SR Ca2+content with voltage clamp in ventricular myocytes isolated from wild type (WT) mice and transgenic (SRTG) mice. The amplitude of [Ca2+]itransients was insignificantly increased in SRTG myocytes, while the diastolic [Ca2+]itended to be lower. The initial and terminal declines of [Ca2+]itransients were significantly accelerated in SRTG myocytes, implying a functional upregulation of the SR CaATPase. We examined the functional contribution of only the SR CaATPase to the initial and the terminal phase of the decline of [Ca2+]i, by abruptly inhibiting Na/Ca exchange with a rapid switcher device. The rate of [Ca2+] decline mediated by the SR CaATPase was increased by 40% in SRTG compared with WT myocytes. The function of the L-type Ca2+channel was unchanged in SRTG myocytes, while INa/Ca density was slightly (10%) decreased. Measured SR Ca2+content was significantly increased by 29% in SRTG myocytes. Thus, overexpression of SR CaATPase markedly accelerates the decline of [Ca2+]itransients, and induces an increase in SR Ca2+content, with some downregulation of the Na/Ca exchanger.

Influence of prior Na+ pump activity on pump and Na+/Ca2+ exchange currents in mouse ventricular myocytes.

We examined the dependence of peak Na+ pump and Na+/Ca2+ exchanger currents on prior Na+ pump inhibition induced by exposure to zero extracellular K+ in voltage-clamped adult murine ventricular myocytes. Abrupt activation of the Na+ pump by reexposure of myocytes to extracellular K+ with a rapid solution switcher resulted in the development of a transient peak current at approximately 500 ms, followed by a decline over 1-2 min to a steady-state level. The magnitudes of both the peak Na+ pump current (Ip) and the peak outward Na+/Ca2+ exchange current, activated by rapidly reducing extracellular Na+ to zero with the solution switcher, were dependent on previous Na+ pump activity. [Na+] gradients (Na+-binding benzofuran isophthalate fluorescence) between the patch pipette and the bulk cytosol were relatively small and could not account for the large differences between peak and steady-state Ip and reverse Na+/Ca2+ exchanger currents. Our results are consistent with the presence of a subsarcolemmal Na+ concentration gradient, which is similar for the Na+ pump and the Na+/Ca2+ exchanger. These findings also support the hypothesis that the Na+ pump and the Na+/Ca2+ exchanger are colocalized in the sarcolemma.

The transient outward current in mice lacking the potassium channel gene Kv1.4.

1. The transient outward current (Ito) plays a prominent role in the repolarization phase of the cardiac action potential. Several K+ channel genes, including Kv1.4, are expressed in the heart, produce rapidly inactivating currents when heterologously expressed, and may be the molecular basis of Ito. 2. We engineered mice homozygous for a targeted disruption of the K+ channel gene Kv1.4 and compared Ito in wild-type (Kv1.4+/+), heterozygous (Kv1.4+/-) and homozygous 'knockout' (Kv1.4-/-) mice. Kv1.4 RNA was truncated in Kv1.4-/- mice and protein expression was absent. 3. Adult myocytes isolated from Kv1.4+/+, Kv1.4+/- and Kv1.4-/- mice had large rapidly inactivating outward currents. The peak current densities at 60 mV (normalized by cellular capacitance, in pA pF-1; means +/- s.e.m.) were 53.8 +/- 5. 3, 45.3 +/- 2.2 and 44.4 +/- 2.8 in cells from Kv1.4+/+, Kv1.4+/- and Kv1.4-/- mice, respectively (P < 0.02 for Kv1.4+/+ vs. Kv1.4-/-). The steady-state values (800 ms after the voltage clamp step) were 30.9 +/- 2.9, 26.9 +/- 3.8 and 23.5 +/- 2.2, respectively (P < 0.02 for Kv1.4+/+ vs. Kv1.4-/-). The inactivating portion of the current was unchanged in the targeted mice. 4. The voltage dependence and time course of inactivation were not changed by targeted disruption of Kv1.4. The mean best-fitting V (membrane potential at 50 % inactivation) values for myocytes from Kv1.4 +/+, Kv1.4+/- and Kv1. 4-/- mice were -53.5 +/- 3.7, -51.1 +/- 2.6 and -54.2 +/- 2.4 mV, respectively. The slope factors (k) were -10.1 +/- 1.4, -8.8 +/- 1.4 and -9.5 +/- 1.2 mV, respectively. The fast time constants for development of inactivation at -30 mV were 27.8 +/- 2.2, 26.2 +/- 5. 1 and 19.6 +/- 2.1 ms in Kv1.4+/+, Kv1.4+/- and Kv1.4-/- myocytes, respectively. At +30 mV, they were 35.5 +/- 2.6, 30.0 +/- 2.1 and 28. 7 +/- 1.6 ms, respectively. The time constants for the rapid phase of recovery from inactivation at -80 mV were 32.5 +/- 8.2, 23.3 +/- 1.8 and 39.0 +/- 3.7 ms, respectively. 5. Nearly the entire inactivating component as well as more than 60 % of the steady-state outward current was eliminated by 1 mM 4-aminopyridine in Kv1.4+/+, Kv1.4+/- and Kv1.4-/- myocytes. 6. Western blot analysis of heart membrane extracts showed no significant upregulation of the Kv4 subfamily of channels in the targeted mice. 7. Thus, Kv1.4 is not the molecular basis of Ito in adult murine ventricular myocytes.