H B Nuss

Ionic Mechanism of Action Potential Prolongation in Ventricular Myocytes From Dogs With Pacing-Induced Heart Failure

Membrane current abnormalities have been described in human heart failure. To determine whether similar current changes are observed in a large animal model of heart failure, we studied dogs with pacing-induced cardiomyopathy. Myocytes isolated from the midmyocardium of 13 dogs with heart failure induced by 3 to 4 weeks of rapid ventricular pacing and from 16 nonpaced control dogs did not differ in cell surface area or resting membrane potential. Nevertheless, action potential duration (APD) was significantly prolonged in myocytes isolated from failing ventricles (APD at 90% repolarization, 1097 +/- 73 milliseconds [failing hearts, n = 30] versus 842 +/- 56 milliseconds [control hearts, n = 25]; P < .05), and the prominent repolarizing notch in phase 1 was dramatically attenuated. Basal L-type Ca2+ current and whole-cell Na+ current did not differ in cells from failing and from control hearts, but significant differences in K+ currents were observed. The density of the inward rectifier K+ current (IKl) was reduced in cells from failing hearts at test potentials below -90 mV (at -150 mV, -19.1 +/- 2.2 pA/pF [failing hearts, n = 18] versus -32.2 +/- 5.1 pA/pF [control hearts, n = 15]; P < .05). The small outward current component of IKl was also reduced in cells from failing hearts (at -60 mV, 1.7 +/- 0.2 pA/pF [failing hearts] versus 2.5 +/- 0.2 pA/pF [control hearts]; P < .05). The peak of the Ca(2+)-independent transient outward current (Ito) was dramatically reduced in myocytes isolated from failing hearts compared with nonfailing control hearts (at +80 mV, 7.0 +/- 0.9 pA/pF [failing hearts, n = 20] versus 20.4 +/- 3.2 pA/pF [control hearts, n = 15]; P < .001), while the steady state component was unchanged. There were no significant differences in Ito kinetics or single-channel conductance. A reduction in the number of functional Ito channels was demonstrated by nonstationary fluctuation analysis (0.4 +/- 0.03 channels per square micrometer [failing hearts, n = 5] versus 1.2 +/- 0.1 channels per square micrometer [control hearts, n = 3]; P < .001). Pharmacological reduction of Ito by 4-aminopyridine in control myocytes decreased the notch amplitude and prolonged the APD. Current clamp-release experiments in which current was injected for 8 milliseconds to reproduce the notch sufficed to shorten the APD significantly in cells from failing hearts. These data support the hypothesis that downregulation of Ito in pacing-induced heart failure is at least partially responsible for the action potential prolongation. Because the repolarization abnormalities mimic those in cells isolated from failing human ventricular myocardium, canine pacing-induced cardiomyopathy may provide insights into the development of repolarization abnormalities and the mechanisms of sudden death in patients with heart failure.

Suppression of Neuronal and Cardiac Transient Outward Currents by Viral Gene Transfer of Dominant-Negative Kv4.2 Constructs

To probe the molecular identity of transient outward (A-type) potassium currents, we expressed a truncated version of Kv4.2 in heart cells and neurons. The rat Kv4.2-coding sequence was truncated at a position just past the first transmembrane segment and subcloned into an adenoviral shuttle vector downstream of a cytomegalovirus promoter (pE1Kv4.2ST). We hypothesized that this construct would act as a dominant-negative suppressor of currents encoded by the Kv4 family by analogy to Kv1 channels. Cotransfection of wild-type Kv4.2 with a β-galactosidase expression vector in Chinese hamster ovary (CHO)-K1 cells produced robust transient outward currents (Ito) after two days (14.0 pA/pF at 50 mV,n = 5). Cotransfection with pE1Kv4.2ST markedly suppressed the Kv4.2 currents (0.8 pA/pF, n = 6,p < 0.02; cDNA ratio of 2:1 Kv4.2ST:wild type), but in parallel experiments, it did not alter the current density of coexpressed Kv1.4 or Kv1.5 channels. Kv4.2ST also effectively suppressed rat Kv4.3 current when coexpressed in CHO-K1 cells. We then engineered a recombinant adenovirus (AdKv4.2ST) designed to overexpress Kv4.2ST in infected cells. A-type currents in rat cerebellar granule cells were decreased two days after AdKv4.2ST infection as compared with those infected by a β-galactosidase reporter virus (116.0 pA/pFversus 281.4 pA/pF in Ad β-galactosidase cells,n = 8 each group, p < 0.001). Likewise, Ito in adult rat ventricular myocytes was suppressed by AdKv4.2ST but not by Adβ-galactosidase (8.8 pA/pFversus 21.4 pA/pF in β-galactosidase cells,n = 6 each group, p < 0.05). Expression of a GFP-Kv4.2ST fusion construct enabled imaging of subcellular protein localization by confocal microscopy. The protein was distributed throughout the surface membrane and intracellular membrane systems. We conclude that genes from the Kv4 family are the predominant contributors to the A-type currents in cerebellar granule cells and Ito in rat ventricle. Overexpression of dominant-negative constructs may be of general utility in dissecting the contributions of various ion channel genes to excitability.

EXTERNAL PORE RESIDUE MEDIATES SLOW INACTIVATION IN MU 1 RAT SKELETAL MUSCLE SODIUM CHANNELS

1. Upon depolarization, voltage‐gated sodium channels assume non‐conducting inactivated states which may be characterized as ‘fast’ or ‘slow’ depending on the length of the repolarization period needed for recovery. Skeletal muscle Na+ channel alpha‐subunits expressed in Xenopus laevis oocytes display anomalous gating behaviour, with substantial slow inactivation after brief depolarizations. We exploited this kinetic behaviour to examine the structural basis for slow inactivation. 2. While fast inactivation in Na+ channels is mediated by cytoplasmic occlusion of the pore by III‐IV linker residues, the structural features of slow inactivation are unknown. Since external pore‐lining residues modulate C‐type inactivation in potassium channels, we performed serial cysteine mutagenesis in the permeation loop (P‐loop) of the rat skeletal muscle Na+ channel (mu 1) to determine whether similarly placed residues are involved in Na+ channel slow inactivation. 3. Wild‐type and mutant alpha‐subunits were heterologously expressed in Xenopus oocytes, and Na+ currents were recorded using a two‐electrode voltage clamp. Slow inactivation after brief depolarizations was eliminated by the W402C mutation in domain I. Cysteine substitution of the homologous tryptophan residues in domains II, III and IV did not alter slow inactivation. 4. Analogous to the W402C mutation, coexpression of the wild‐type alpha‐subunit with rat brain Na+ channel beta 1‐subunit attenuated slow inactivation. However, the W402C mutation imposed a delay on recovery from fast inactivation, while beta 1‐subunit coexpression did not. We propose that the W402C mutation and the beta 1‐subunit modulate gating through distinct mechanisms. 5. Removal of fast inactivation in wild‐type alpha‐subunits with the III‐IV linker mutation I1303Q; F1304Q; M1305Q markedly slowed the development of slow inactivation. We propose that slow inactivation in Na+ channels involves conformational changes in the external pore. Mutations that affect fast and slow inactivation appear to interact despite their remote positions in the channel.

Overexpression of a human potassium channel suppresses cardiac hyperexcitability in rabbit ventricular myocytes

The high incidence of sudden death in heart failure may reflect abnormalities of repolarization and heightened susceptibility to arrhythmogenic early afterdepolarizations (EADs). We hypothesized that overexpression of the human K+ channel HERG (human ether-a-go-go-related gene) could enhance repolarization and suppress EADs. Adult rabbit ventricular myocytes were maintained in primary culture, which suffices to prolong action potentials and predisposes to EADs. To achieve efficient gene transfer, we created AdHERG, a recombinant adenovirus containing the HERG gene driven by a Rous sarcoma virus (RSV) promoter. The virally expressed HERG current exhibited pharmacologic and kinetic properties like those of native IKr. Transient outward currents in AdHERG-infected myocytes were similar in magnitude to those in control cells, while stimulated action potentials (0.2 Hz, 37 degrees C) were abbreviated compared with controls. The occurrence of EADs during a train of action potentials was reduced by more than fourfold, and the relative refractory period was increased in AdHERG-infected myocytes compared with control cells. Gene transfer of delayed rectifier potassium channels represents a novel and effective strategy to suppress arrhythmias caused by unstable repolarization.

Local anesthetics as effectors of allosteric gating. Lidocaine effects on inactivation-deficient rat skeletal muscle Na channels.

Time- and voltage-dependent local anesthetic effects on sodium (Na) currents are generally interpreted using modulated receptor models that require formation of drug-associated nonconducting states with high affinity for the inactivated channel. The availability of inactivation-deficient Na channels has enabled us to test this traditional view of the drug-channel interaction. Rat skeletal muscle Na channels were mutated in the III-IV linker to disable fast inactivation (F1304Q: FQ). Lidocaine accelerated the decay of whole-cell FQ currents in Xenopus oocytes, reestablishing the wild-type phenotype; peak inward current at -20 mV was blocked with an IC50 of 513 microM, while plateau current was blocked with an IC50 of only 74 microM (P < 0.005 vs. peak). In single-channel experiments, mean open time was unaltered and unitary current was only reduced at higher drug concentrations, suggesting that open-channel block does not explain the effect of lidocaine on FQ plateau current. We considered a simple model in which lidocaine reduced the free energy for inactivation, causing altered coupling between activation and inactivation. This model readily simulated macroscopic Na current kinetics over a range of lidocaine concentrations. Traditional modulated receptor models which did not modify coupling between gating processes could not reproduce the effects of lidocaine with rate constants constrained by single-channel data. Our results support a reinterpretation of local anesthetic action whereby lidocaine functions as an allosteric effector to enhance Na channel inactivation.

Adenovirus-mediated expression of a voltage-gated potassium channel in vitro (rat cardiac myocytes) and in vivo (rat liver). A novel strategy for modifying excitability.

Excitability is governed primarily by the complement of ion channels in the cell membrane that shape the contour of the action potential. To modify excitability by gene transfer, we created a recombinant adenovirus designed to overexpress a Drosophila Shaker potassium channel (AdShK). In vitro, a variety of mammalian cell types infected with AdShK demonstrated robust expression of the exogenous channel. Spontaneous action potentials recorded from cardiac myocytes in primary culture were abbreviated compared with noninfected myocytes. Intravascular infusion of AdShK in neonatal rats induced Shaker potassium channel mRNA expression in the liver, and large potassium currents could be recorded from explanted hepatocytes. Thus, recombinant adenovirus technology has been used for in vitro and in vivo gene transfer of ion channel genes designed to modify cellular action potentials. With appropriate targeting, such a strategy may be useful in gene therapy of arrhythmias, seizure disorders, and myotonic muscle diseases.

Coupling between fast and slow inactivation revealed by analysis of a point mutation (F1304Q) in mu 1 rat skeletal muscle sodium channels.

1. We sought to elucidate the mechanism of the defective inactivation that characterizes sodium channels containing mutations in the cytoplasmic loop between the third and fourth domains (the III‐IV linker). Specifically, we measured whole‐cell and single‐channel currents through wild‐type and F1304Q mutant mu 1 rat skeletal muscle Na+ channels expressed in Xenopus laevis oocytes. 2. In wild‐type channels, inactivation is complete and the faster of two decay components predominates. In F1304Q, inactivation is incomplete; the slow decay component is larger in amplitude and slower than in wild‐type. The fraction of non‐inactivating current is substantial (37 +/‐ 2% of peak current at ‐20 mV) in F1304Q. 3. Cell‐attached patch recordings confirmed the profound kinetic differences and indicated that permeation was not altered by the F1304Q mutation. The F1304Q phenotype must be conferred entirely by changes in gating properties and is not remedied by coexpression with the beta 1‐subunit. 4. Recovery from inactivation of F1304Q channels is faster than for wild‐type channels and three exponentials are required to describe recovery adequately following long (5 s) depolarizations. Thus, there are three inactivated states even in ‘inactivation‐deficient’ F1304Q channels. 5. The steady‐state voltage dependence of F1304Q inactivation is right‐shifted by 26 +/‐ 2 mV. 6. A gating model incorporating three inactivated states, all directly accessible from multiple closed states or the open state, was constrained to fit wild‐type and F1304Q inactivation (h infinitive) data and repriming data simultaneously. While it was necessary to alter the rate constants entering and exiting all three inactivated states, the model accounted for the F1304Q‐induced rightward shift in steady‐state inactivation without imposing voltage dependence on the inactivation rate constants. 7. We conclude that the F1304Q mutation in mu 1 sodium channels modifies several inactivation processes simultaneously. The fact that a single amino acid substitution profoundly alters both fast and slow inactivation indicates that these processes share physical determinants in Na+ channels.

Prospects for Genetic Manipulation of Cardiac Excitability

Despite impressive advances in the therapy of a number of types of heart disease in the last two decades, sudden cardiac death remains a public health problem of staggering dimensions. Current treatment options include antiarrhythmic drugs that have higher than desired failure rates and implantable defibrillators that incur significant costs to the patient and society. The development of therapies that better suppress the cardiac arrhythmias responsible for sudden cardiac death requires a broad and comprehensive understanding of the basic mechanisms underlying electrical instability in the heart. This study explores the scientific basis for a molecular genetic approach to modify cardiac excitability and thereby to create animal models of sudden cardiac death. The availability of such models will open up new avenues of research in arrhythmogenesis and facilitate the development of novel antiarrhythmic agents.