Adult Mouse Ventricle Research Articles

KChIP2 modulates the cell surface expression of Kv1.5-encoded K + channels

The Kv channel interacting proteins (KChIPs) were identified in a yeast two hybrid screen using the N terminus of Kv4.3 as bait. Previous studies have demonstrated that KChIP2 associates with voltage-gated K + (Kv) pore-forming (α) subunits of the Kv4 subfamily and contributes to the formation of the rapidly inactivating and recovering Kv4-encoded cardiac transient outward K + channels, I to,f. Here, we report that co-expression of KChIP2 (or KChIP1) also modulates the functional cell surface expression of Kv1.5-encoded K + channels in transiently transfected HEK-293 cells. In contrast to the effects of KChIP2 on Kv4 channels, however, co-expression of KChIP2 (or KChIP1) decreases Kv1.5-encoded K + currents. Although current densities are reduced, KChIP2 (or KChIP1) co-expression does not affect the time- or voltage-dependent properties of heterologously expressed Kv1.5-encoded K + currents. Immunohistochemical and cell surface biotinylation experiments demonstrate that KChIP2 reduces the cell surface expression of Kv1.5, likely by inhibiting forward trafficking from the endoplasmic reticulum. In addition, biochemical experiments reveal that KChIP2 co-immunoprecipitates with Kv1.5 (as well as Kv4.2/Kv4.3) from adult mouse ventricles, demonstrating that, similar to other Kv accessory subunits, KChIP2 is a multifunctional Kv channel accessory subunit. Taken together, the results here suggest that KChIP2 contributes to the formation of functional mouse ventricular (Kv1.5-encoded) I K,slow1 channels as well, perhaps, as other Kv1.5-encoded K + currents, including I Kur ( I K,ultrarapid), in human atria.

Developmental expression of NCS-1 (frequenin), a regulator of Kv4 K+ channels, in mouse heart.

The channel proteins responsible for the cardiac transient outward K+ current (Ito) of human and rodent heart are composed, in part, of pore-forming Kv4.3 or Kv4.2 principal subunits. Recent reports implicate K+ channel interacting proteins (members of the neuronal Ca2+-binding protein family) as subunits of the Ito channel complex. We reported that another Ca2+-binding protein, frequenin [or neuronal calcium center protein-1 (NCS-1)], also functions as a Kv4 auxiliary subunit in the brain. By examining cardiac expression of NCS-1, the aim of this study was to examine the potential physiologic relevance of this protein as an additional regulator of cardiac Ito. Immunoblot analysis demonstrates NCS-1 protein to be expressed in adult mouse ventricle at levels comparable to that found in some brain regions. Cardiac NCS-1 protein expression levels are much higher in fetal and neonatal mouse hearts when compared with the adult. Immunocytochemical analysis of isolated neonatal mouse ventricular myocytes demonstrates co-localization of NCS-1 and Kv4.2 proteins at the sarcolemma. Given its high levels of expression in the heart, NCS-1 should be considered an important potential Kv4 regulatory subunit, particularly in the immature heart.

Pharmacological evidence for involvement of phospholipase D, protein kinase C, and sodium-calcium exchanger in alpha-adrenoceptor-mediated negative inotropy in adult mouse ventricle.

The intracellular signalling pathway for alpha-adrenoceptor-mediated negative inotropy was studied pharmacologically in isolated adult mouse ventricle. The negative inotropy was inhibited by GF-109203X, a nonselective protein kinase C inhibitor. Phorbol 12-myristate 13-acetate also produced sustained negative inotropy, which was inhibited by KB-R7943, a Na(+)/Ca(2+) exchanger inhibitor. The alpha-adrenoceptor-mediated negative inotropy was augmented by RHC-80267, a diacylglycerol lipase inhibitor, but was inhibited either by C(2)-ceramide, a phospholipase D inhibitor, and high concentration of propranolol (50 micro M), which inhibits phosphatidate phosphohydrolase. The inotropy was not affected by U-73122, a phospholipase C inhibitor. Lavendustin-A, a tyrosine kinase inhibitor, also inhibited the negative inotropy. These findings suggest that alpha-adrenoceptor-mediated negative inotropy in adult mouse ventricle is mediated by activation of tyrosine kinase, the phospholipase D-phosphatidate phosphohydrolase pathway, and protein kinase C.

Pharmacological Evidence for Involvement of Phospholipase D, Protein Kinase C, and Sodium-Calcium Exchanger in -Adrenoceptor-Mediated Negative Inotropy in Adult Mouse Ventricle

Pharmacological Evidence for Involvement of Phospholipase D, Protein Kinase C, and Sodium-Calcium Exchanger in -Adrenoceptor-Mediated Negative Inotropy in Adult Mouse Ventricle

Microarray analysis reveals complex remodeling of cardiac ion channel expression with altered thyroid status: relation to cellular and integrated electrophysiology.

Although electrophysiological remodeling occurs in various myocardial diseases, the underlying molecular mechanisms are poorly understood. cDNA microarrays containing probes for a large population of mouse genes encoding ion channel subunits ("IonChips") were developed and exploited to investigate remodeling of ion channel transcripts associated with altered thyroid status in adult mouse ventricle. Functional consequences of hypo- and hyperthyroidism were evaluated with patch-clamp and ECG recordings. Hypothyroidism decreased heart rate and prolonged QTc duration. Opposite changes were observed in hyperthyroidism. Microarray analysis revealed that hypothyroidism induces significant reductions in KCNA5, KCNB1, KCND2, and KCNK2 transcripts, whereas KCNQ1 and KCNE1 expression is increased. In hyperthyroidism, in contrast, KCNA5 and KCNB1 expression is increased and KCNQ1 and KCNE1 expression is decreased. Real-time RT-PCR validated these results. Consistent with microarray analysis, Western blot experiments confirmed those modifications at the protein level. Patch-clamp recordings revealed significant reductions in I(to,f) and I(K,slow) densities, and increased I(Ks) density in hypothyroid myocytes. In addition to effects on K+ channel transcripts, transcripts for the pacemaker channel HCN2 were decreased and those encoding the alpha1C Ca2+ channel (CaCNA1C) were increased in hypothyroid animals. The expression of Na+, Cl-, and inwardly rectifying K+ channel subunits, in contrast, were unaffected by thyroid hormone status. Taken together, these data demonstrate that thyroid hormone levels selectively and differentially regulate transcript expression for at least nine ion channel alpha- and beta-subunits. Our results also document the potential of cDNA microarray analysis for the simultaneous examination of ion channel transcript expression levels in the diseased/remodeled myocardium.

Modulation of Kv4-encoded K+ Currents in the Mammalian Myocardium by Neuronal Calcium Sensor-1

Voltage-gated K(+) channels are multimeric proteins, consisting of four pore-forming alpha-subunits alone or in association with accessory subunits. Recently, for example, it was shown that the accessory Kv channel interacting proteins form complexes with Kv4 alpha-subunits and modulate Kv4 channel activity. The experiments reported here demonstrate that the neuronal calcium sensor protein-1 (NCS-1), another member of the recoverin-neuronal calcium sensor superfamily, is expressed in adult mouse ventricles and that NCS-1 co-immunoprecipitates with Kv4.3 from (adult mouse) ventricular extracts. In addition, co-expression studies in HEK-293 cells reveal that NCS-1 increases membrane expression of Kv4 alpha-subunits and functional Kv4-encoded K(+) current densities. Co-expression of NCS-1 also decreases the rate of inactivation of Kv4 alpha-subunit-encoded K(+) currents. In contrast to the pronounced effects of Kv channel interacting proteins on Kv4 channel gating, however, NCS-1 co-expression does not measurably affect the voltage dependence of steady-state inactivation or the rate of recovery from inactivation of Kv4-encoded K(+) currents. Taken together, these results suggest that NCS-1 is an accessory subunit of Kv4-encoded I(to,f) channels that functions to regulate I(to,f) density in the mammalian myocardium.

Role of heteromultimers in the generation of myocardial transient outward K+ currents.

Previous studies have demonstrated a role for Kv4 alpha subunits in the generation of the fast transient outward K+ current, I(to,f), in the mammalian myocardium. The experiments here were undertaken to explore the role of homomeric/heteromeric assembly of Kv4.2 and Kv4.3 and of the Kv channel accessory subunit, KChIP2, in the generation of mouse ventricular I(to,f). Western blots reveal that the expression of Kv4.2 parallels the regional heterogeneity in I(to,f) density, whereas Kv4.3 and KChIP2 are uniformly expressed in adult mouse ventricles. Antisense oligodeoxynucleotides (AsODNs) targeted against Kv4.2 or Kv4.3 selectively attenuate I(to,f) in mouse ventricular cells. Adenoviral-mediated coexpression of Kv4.2 and Kv4.3 in HEK-293 cells and in mouse ventricular myocytes produces transient outward K+ currents with properties distinct from those produced on expression of Kv4.2 or Kv4.3 alone, and the gating properties of the heteromeric Kv4.2/Kv4.3 channels in ventricular cells are more similar to native I(to,f) than are the homomeric Kv4.2 or Kv4.3 channels. Biochemical studies reveal that Kv4.2, Kv4.3, and KChIP2 coimmunoprecipitate from adult mouse ventricles. In addition, most of the Kv4.2 and KChIP2 are associated with Kv4.3 in situ. Taken together, these results demonstrate that functional mouse ventricular I(to,f) channels are heteromeric, comprising Kv4.2/Kv4.3 alpha subunits and KChIP2. The results here also suggest that Kv4.2 is the primary determinant of the regional heterogeneity in I(to,f) expression in adult mouse ventricle.

Expression of the beta (slow)-isoform of MHC in the adult mouse heart causes dominant-negative functional effects.

Alpha- and beta-myosin heavy chain (MHC), the two MHC isoforms expressed in the mammalian heart, differ quantitatively in their enzymatic activities. The MHC composition of the heart can change dramatically in response to numerous stimuli, leading to the hypothesis that changes in cardiac function can be caused by myosin isoform shifts. However, this hypothesis has remained unproven because the stimuli used to generate these shifts are complex and accompanied by many additional physiological changes, including alterations in cardiac mass and geometry. Adult mouse ventricles normally express only alpha-MHC (the faster motor). To determine whether genetic alteration of the MHC isoform composition in the adult mouse heart would result in changes in cardiac chamber mass and contractility, we established transgenic mouse lines that express a Myc-tagged beta-MHC molecule (the slower motor) in adult ventricular tissue, one of which expresses 12% of its myosin as the transgene. There is no evidence of hypertrophy, induction of hypertrophic markers, and no histopathology. Myofibrillar Ca(2+)-activated ATPase activity is decreased by 23%, and Langendorff preparations demonstrate a significant 15% decrease in systolic function in transgenic hearts. These results suggest that even small shifts in the myosin isoform composition of the myocardium can result in physiologically significant changes in cardiac contractility and could be relevant to cardiovascular disease.

Molecular basis of transient outward K+ current diversity in mouse ventricular myocytes.

1. Two kinetically and pharmacologically distinct transient outward K+ currents, referred to as Ito,f and Ito,s, have been distinguished in mouse left ventricular myocytes. Ito,f is present in all left ventricular apex cells and in most left ventricular septum cells, whereas Ito,s is identified exclusively in left ventricular septum cells. 2. Electrophysiological recordings from ventricular myocytes isolated from animals with a targeted deletion of the Kv1.4 gene (Kv1.4-/- mice) reveal that Ito,s is undetectable in cells isolated from the left ventricular septum (n = 26). Ito,f density in both apex and septum cells, in contrast, is not affected by deletion of Kv1.4. 3. Neither the 4-AP-sensitive, slowly inactivating K+ current, IK,slow, nor the steady-state non-inactivating K+ current, ISS, is affected in Kv1.4-/- mouse left ventricular cells. 4. In myocytes isolated from transgenic mice expressing a dominant negative Kv4.2 alpha subunit, Kv4.2W362F, Ito,f is eliminated in both left ventricular apex and septum cells. In addition, a slowly inactivating transient outward K+ current similar to Ito,s in wild-type septum cells is evident in myocytes isolated from left ventricular apex of Kv4.2W362F-expressing transgenics. The density of Ito,s in septum cells, however, is unaffected by Kv4.2W362F expression. 5. Western blots of fractionated mouse ventricular membrane proteins reveal a significant increase in Kv1.4 protein level in Kv4.2W362F-expressing transgenic mice. The protein levels of other Kv alpha subunits, Kv1.2 and Kv2.1, in contrast, are not affected by the expression of the Kv4.2W362F transgene. 6. The results presented here demonstrate that the molecular correlates of Ito,f and Ito,s in adult mouse ventricle are distinct. Kv1.4 underlies mouse ventricular septum Ito,s, whereas Kv alpha subunits of the Kv4 subfamily underlie mouse ventricular apex and septum Ito, f. The appearance of the slow transient outward K+ current in Kv4. 2W362F-expressing left ventricular apex cells with properties indistinguishable from Ito,s in wild-type cells is accompanied by an increase in Kv1.4 protein expression, suggesting that the upregulation of Kv1.4 underlies the observed electrical remodeling in Kv4.2W362F-expressing transgenics.

Inhibition of cardiac delayed rectifier K+ current by overexpression of the long-QT syndrome HERG G628S mutation in transgenic mice.

Mutations in the HERG gene are linked to the LQT2 form of the inherited long-QT syndrome. Transgenic mice were generated expressing high myocardial levels of a particularly severe form of LQT2-associated HERG mutation (G628S). Hearts from G628S mice appeared normal except for a modest enlargement seen only in females. Ventricular myocytes isolated from adult wild-type hearts consistently exhibited an inwardly rectifying E-4031-sensitive K+ current resembling the rapidly activating cardiac delayed rectifier K+ current (Ikr) in its time and voltage dependence; this current was not found in cells isolated from G628S mice. Action potential duration was significantly prolonged in single myocytes from G628S ventricle (cycle length=1 second, 26 degrees C) but not in recordings from intact ventricular strips studied at more physiological rates and temperature (200 to 400 bpm, 37 degrees C). ECG intervals, including QT duration, were unchanged, although minor aberrancies were noted in 20% (16/80) of the G628S mice studied, primarily involving the QRS complex and, more rarely, T-wave morphology. The aberrations were more commonly observed in females than males but could not be correlated with sex-based differences in action potential duration. These results establish the presence of IKr in the adult mouse ventricle and demonstrate the ability of the G628S mutation to exert a dominant negative effect on endogenous IKr in vivo, leading to the expected LQT2 phenotype of prolonged repolarization at the single cell level but not QT prolongation in the intact animal. The model may be useful in dissecting repolarization currents in the mouse heart and as a means of examining the mechanism(s) by which the G628S mutation exerts its dominant negative effect on native cardiac cells in vivo.

A rapidly activating sustained K+ current modulates repolarization and excitation-contraction coupling in adult mouse ventricle.

1. The K+ currents which control repolarization in adult mouse ventricle, and the effects of changes in action potential duration on excitation-contraction coupling in this tissue, have been studied with electrophysiological methods using single cell preparations and by recording mechanical parameters from an in vitro working heart preparation. 2. Under conditions where Ca(2+)-dependent currents were eliminated by buffering intracellular Ca2+ with EGTA, depolarizing voltage steps elicited two rapidly activating outward K+ currents: (i) a transient outward current, and (ii) a slowly inactivating or 'sustained' delayed rectifier. 3. These two currents were separated pharmacologically by the K+ channel blocker 4-amino-pyridine (4-AP). 4-AP at concentrations between 3 and 200 microM resulted in (i) a marked increase in action potential duration and a large decrease in the sustained K+ current at plateau potentials, as well as (ii) a significant increase in left ventricular systolic pressure in the working heart preparation. 4. The current-voltage (I-V) relation, kinetics, and block by low concentrations of 4-AP strongly suggest that the rapid delayed rectifier in adult mouse ventricles is the same K+ current (Kv1.5) that has been characterized in detail in human and canine atria. 5. These results show that the 4-AP-sensitive rapid delayed rectifier is a very important repolarizing current in mouse ventricle. The enhanced contractility produced by 4-AP (50 microM) in the working heart preparation demonstrates that modulation of the action potential duration, by blocking a K+ current, is a very significant inotropic variable.

Developmental changes in the delayed rectifier K+ channels in mouse heart.

Expression of cardiac transient outward current and inwardly rectifying K+ current is age dependent. However, little is known about age-related changes in cardiac delayed rectifier K+ current (IK, with rapidly and slowly activating components, IKr and IKs, respectively). Accordingly, the purpose of the present study was to assess developmental changes in IK channels in fetal, neonatal, and adult mouse ventricles. Three techniques were used: conventional microelectrode to measure the action potential, voltage clamp to record macroscopic currents of IK, and radioligand assay to examine [3H]dofetilide binding sites. The extent of prolongation of action potential duration at 95% repolarization (APD95) by a selective IKr blocker, dofetilide (1 mumol/L), dramatically decreased from fetal (137% +/- 18%) to day-1 (75% +/- 29%) and day-3 (20% +/- 15%) neonatal mouse ventricular tissues (P < .01). Dofetilide did not prolong APD95 in adult myocardium. IKr is the sole component of IK in day-18 fetal mouse ventricular myocytes. However, both IKr and IKs were observed in day-1 neonatal ventricular myocytes. With further development, IKs became the dominant component of IK in day-3 neonates. In adult mouse ventricular myocytes, neither IKr nor IKs was observed. Correspondingly, a high-affinity binding site for [3H]dofetilide was present in fetal mouse ventricles but was absent in adult ventricles. The complementary data from microelectrode, voltage-clamp, and [3H]dofetilide binding studies demonstrate that expression of the IK channel is developmentally regulated in the mouse heart.