In the experiments here, the time- and voltage-dependent properties of the Ca2+-independent, depolarization-activated K+ currents in adult mouse ventricular myocytes were characterized in detail. In the majority (65 of 72, ≈ 90%) of cells dispersed from the ventricles, analysis of the decay phases of the outward currents revealed three distinct K+ current components: a rapidly inactivating, transient outward K+ current, Ito,f (mean ± SEM τdecay = 85 ± 2 ms); a slowly (mean ± SEM τdecay = 1,162 ± 29 ms) inactivating K+ current, IK,slow; and a non inactivating, steady state current, Iss. In a small subset (7 of 72, ≈ 10%) of cells, Ito,f was absent and a slowly inactivating (mean ± SEM τdecay = 196 ± 7 ms) transient outward current, referred to as Ito,s, was identified; the densities and properties of IK,slow and Iss in Ito,s-expressing cells are indistinguishable from the corresponding currents in cells with Ito,f. Microdissection techniques were used to remove tissue pieces from the left ventricular apex and from the ventricular septum to allow the hypothesis that there are regional differences in Ito,f and Ito,s expression to be tested directly. Electrophysiological recordings revealed that all cells isolated from the apex express Ito,f (n = 35); Ito,s is not detected in these cells (n = 35). In the septum, by contrast, all of the cells express Ito,s (n = 28) and in the majority (22 of 28, 80%) of cells, Ito,f is also present. The density of Ito,f (mean ± SEM at +40 mV = 6.8 ± 0.5 pA/pF, n = 22) in septum cells, however, is significantly (P < 0.001) lower than Ito,f density in cells from the apex (mean ± SEM at +40 mV = 34.6 ± 2.6 pA/pF, n = 35). In addition to differences in inactivation kinetics, Ito,f, Ito,s, and IK,slow display distinct rates of recovery (from inactivation), as well as differential sensitivities to 4-aminopyridine (4-AP), tetraethylammonium (TEA), and Heteropoda toxin-3. IK,slow, for example, is blocked selectively by low (10–50 μM) concentrations of 4-AP and by (≥25 mM) TEA. Although both Ito,f and Ito,s are blocked by high (>100 μM) 4-AP concentrations and are relatively insensitive to TEA, Ito,f is selectively blocked by nanomolar concentrations of Heteropoda toxin-3, and Ito,s (as well as IK,slow and Iss) is unaffected. Iss is partially blocked by high concentrations of 4-AP or TEA. The functional implications of the distinct properties and expression patterns of Ito,f and Ito,s, as well as the likely molecular correlates of these (and the IK,slow and Iss) currents, are discussed.

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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. Electrophysiological recordings from ventricular myocytes isolated from animals with a targeted deletion of the Kv1.4gene (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. 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. In myocytes isolated from transgenic mice expressing a dominant negative Kv4.2 α 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. 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 α subunits, Kv1.2 and Kv2.1, in contrast, are not affected by the expression of the Kv4.2W362F transgene. 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 α 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.

An in vivo experimental strategy, involving cardiac-specific expression of a mutant Kv 2.1 subunit that functions as a dominant negative, was exploited in studies focused on exploring the role of members of the Kv2 subfamily of pore-forming (alpha) subunits in the generation of functional voltage-gated K(+) channels in the mammalian heart. A mutant Kv2.1 alpha subunit (Kv2.1N216) was designed to produce a truncated protein containing the intracellular N terminus, the S1 membrane-spanning domain, and a portion of the S1/S2 loop. The truncated Kv2.1N216 was epitope tagged at the C terminus with the 8-amino acid FLAG peptide to generate Kv2. 1N216FLAG. No ionic currents are detected on expression of Kv2. 1N216FLAG in HEK-293 cells, although coexpression of this construct with wild-type Kv2.1 markedly reduced the amplitudes of Kv2. 1-induced currents. Using the alpha-myosin heavy chain promoter to direct cardiac specific expression of the transgene, 2 lines of Kv2. 1N216FLAG-expressing transgenic mice were generated. Electrophysiological recordings from ventricular myocytes isolated from these animals revealed that I(K, slow) is selectively reduced. The attenuation of I(K, slow) is accompanied by marked action potential prolongation, and, occasionally, spontaneous triggered activity (apparently induced by early afterdepolarizations) is observed. The time constant of inactivation of I(K, slow) in Kv2. 1N216FLAG-expressing cells (mean+/-SEM=830+/-103 ms; n=17) is accelerated compared with the time constant of I(K, slow) inactivation (mean+/-SEM=1147+/-57 ms; n=25) in nontransgenic cells. In addition, unlike I(K, slow) in wild-type cells, the component of I(K, slow) remaining in the Kv2.1N216FLAG-expressing cells is insensitive to 25 mmol/L tetraethylammonium. Taken together, these observations suggest that there are 2 distinct components of I(K, slow) in mouse ventricular myocytes and that Kv2 alpha subunits underlie the more slowly inactivating, tetraethylammonium-sensitive component of I(K, slow). In vivo telemetric recordings also reveal marked QT prolongation, consistent with a defect in ventricular repolarization, in Kv2.1N216FLAG-expressing transgenic mice.

Abstract-Previous studies have demonstrated a role for voltage-gated K ϩ (Kv) channel ␣ subunits of the Kv4 subfamily in the generation of rapidly inactivating/recovering cardiac transient outward K ϩ current, I to,f , channels. Biochemical studies suggest that mouse ventricular I to,f channels reflect the heteromeric assembly of Kv4.2 and Kv4.3 with the accessory subunits, KChIP2 and Kv␤1, and that Kv4.2 is the primary determinant of regional differences in (mouse ventricular) I to,f densities. Interestingly, the phenotypic consequences of manipulating I to,f expression in different mouse models are distinct. In the experiments here, the effects of the targeted deletion of Kv4. 1 Considerable progress has been made in characterizing the properties of myocardial Kv channels and in defining the roles of individual Kv channel pore-forming (␣) and accessory (␤) subunits in the generation of these channels. 1 In adult mouse ventricles, for example, multiple Kv currents are coexpressed. 2-10 All available evidence suggests that ␣ subunits of the Kv4 subfamily underlie fast inactivating and recovering cardiac transient outward, I to,f , channels. 1 Biochemical studies suggest that mouse ventricular I to,f channels reflect the heteromeric assembly of Kv4.2 and Kv4.3. 10 In large mammals, however, Kv4.2 is not expressed, and I to,f channels are thought to reflect Kv4.3 homotetramers. 11,12 Multiple splice variants of Kv4.3 have been identified, 12 although the role(s) of these variants in the generation of I to,f channels is unclear.The accessory subunit, KChIP2, 13 coimmunoprecipitates with Kv4.2 and Kv4.3 from adult mouse ventricles, 10 and it has been reported that I to,f is eliminated in ventricular myocytes isolated from mice in which the KChIP2 locus was disrupted. 14 In canine ventricles, KChIP2 message 15,16 and protein 17 expression parallel variations in I to,f densities, suggesting that KChIP2 is the primary determinant of I to,f gradients. 15,17 In rodent ventricles, however, KChIP2 is uniformly expressed, and regional differences in I to,f densities are correlated with heterogeneities in Kv4.2 expression. 10,18 Interestingly, the phenotypic consequences of manipulating I to,f expression in vivo are distinct. 4,7,14,19 Recordings from ventricular myocytes isolated from transgenic mice expressing a pore mutant of Kv4.2, Kv4.2W362F, that functions as a dominant negative (Kv4.2DN), for example, revealed that I to,f is eliminated. 4 Materials and MethodsAnimals were handled in accordance with the NIH Guide for the Care and Use of Laboratory Animals; all protocols were approved by the Washington University Animal Studies Committee. The generation of the Kv4.2 Ϫ/Ϫ mice and the methods/protocols used in the present study are detailed in the online data supplement available at http://circres.ahajournals.org. Results Targeted Disruption of the KCND2 (Kv4.2) LocusIn the targeting construct used to generate Kv4.2 Ϫ/Ϫ mice ( Figure 1A), described in the expanded Materials and Methods section in the online data supp...

Abstract-Previous studies have demonstrated a role for Kv4 ␣ subunits in the generation of the fast transient outward K ϩ 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)

Abstract-Effective treatment of systolic hypertension in elderly patients remains a major therapeutic challenge. A multicenter, double-blind, randomized controlled trial with sacubitril/valsartan (LCZ696), a first-in-class angiotensin receptor neprilysin inhibitor, was conducted to determine its effects versus olmesartan (angiotensin receptor blocker) on central aortic pressures, in elderly patients (aged ≥60 years) with systolic hypertension and pulse pressure >60 mm Hg, indicative of arterial stiffness. Patients (n=454; mean age, 67.7 years; mean seated systolic blood pressure, 158.6 mm Hg; mean seated pulse pressure, 69.7 mm Hg) were randomized to receive once-daily sacubitril/valsartan 200 mg or olmesartan 20 mg, force titrated to double the initial doses after 4 weeks, before primary assessment at 12 weeks. The study extended double-blind treatment for 12 to 52 weeks, during which amlodipine (2.5-5 mg) and subsequently hydrochlorothiazide (6.25-25 mg) were added-on for patients not achieving blood pressure target (<140/90

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