Reduced Transient Outward K+ Current and Cardiac Hypertrophy
Causal Relationship or Epiphenomenon?
Action potentials coordinate contraction and relaxation of the heart. The long plateau phase of the action potential ensures sufficient Ca2+ entry into the cytoplasm via L-type Ca2+ channels to invoke Ca2+-induced Ca2+ release from the sarcoplasmic reticulum and activation of the contractile filaments. Action potentials are initiated by opening of voltage-gated Na+ channels that in turn rapidly depolarize the membrane and open voltage-gated Ca2+ and K+ channels. Some K+ channels activate immediately in response to depolarization, then inactivate within tens of milliseconds later, and mediate a current called the transient outward K+ current (Ito). In many mammalian hearts, including human, Ito is responsible for the initial rapid phase of action potential repolarization, discernible as a notch preceding the plateau phase, but has little role in terminal repolarization. In contrast, Ito in rodents is a major repolarizing current throughout the comparatively short cardiac action potential (Figure, panel A) necessary to maintain extremely high heart rates. Ito is composed of at least two components in the mammalian ventricle. One component is characterized by a slow recovery from inactivation (Itos), another by a relatively fast recovery from inactivation (Itof). Comparison of native currents and heterologously expressed channels indicates that Itof is mediated by Kv4.2 and/or Kv4.3 channels, whereas Itos is mediated by Kv1.4 channels.
Prolongation of ventricular action potentials is a common finding in many cardiac disorders, including pathological hypertrophy, heart failure, myocardial infarction, and long-QT syndromes. Two of these diseases, hypertrophy and heart failure, are associated with a decrease of Ito in the human heart and in animal models. Without experimental data to address the question, it might seem reasonable that pathological hypertrophy and heart failure somehow cause a reduction in Ito rather than the opposite. Recently, the relationship between Ito and disease was directly examined with Kv4 and Kv1.4 knockout mouse models.
In 1999, Wickenden et al1 reported the surprising finding that cardiac-specific (driven by α-MHC promoter) overexpression of a dominant-negative Kv4.2 K+ channel subunit in mice caused dilated cardiomyopathy and heart failure, in addition to prolongation of action potential duration (APD). This finding was provocative because an earlier study by Barry et al2 reported that cardiac-specific functional knockout of Itof in mice did not induce hypertrophy. Several differences between the two studies might explain why reduction of Itof in one model but not the other resulted in heart failure. In the earlier study, Barry et al2 overexpressed a Kv4.2 channel subunit that had a single missense mutation (W362F) located in the pore region. In a heterologous expression system, these mutant subunits can fold, coassemble into tetramers, and insert into the plasma membrane normally, but the channels are nonconducting. However, when coexpressed with wild-type Kv4.2 channel subunits, the mutant subunits have a dominant-negative effect, meaning they coassemble with wild-type subunits to form nonfunctional tetrameric channels. In contrast, Wickenden et al1 functionally knocked out Itof by overexpressing an N-terminal fragment of Kv4.2 channel subunits. This partial channel subunit would, of course, have no channel function on its own but could coassemble with wild-type Kv4.2 subunits and have a dominant-negative effect. It is possible that overexpression of the N-terminal fragment of Kv4.2 has toxic effects leading to heart failure. Barry et al2 also noted that knockout of Itof was associated with an increase in Kv1.4 channel expression, perhaps as a compensatory response. However, upregulation of Kv1.4 channel expression was not responsible for the lack of hypertrophy or failure in mice overexpressing Kv4.2W362F because crossbreeding of Kv1.4−/− and Kv4.2W362F transgenic mice produced offspring that had structurally normal hearts despite significant QT prolongation and frequent ventricular tachycardia.3
KChIP2 (Kv channel interacting protein 2) is an auxiliary K+ channel subunit that binds to the N-terminal of Kv4 channel subunits in the heart and increases the number of channels in the plasma membrane and the rate of Kv4 channel recovery from inactivation.4 Regional differences in the expression of KChIP2 are responsible for the transmural gradient in Itof density in the canine heart.5 Recently, Kuo et al6 found that knockout of KChIP in mice nearly eliminated Ito and increased their susceptibility to arrhythmia but did not cause any changes in heart volume, ventricular wall thickness, or contractility.
The KChIP study and most of the Ito knockout mice studies demonstrate that a decrease in Ito does not necessarily result in cellular hypertrophy or heart failure. Conversely, and contrary to most reports, hypertrophy is also not always associated with a change in Ito. Wang et al7 reported in 2001 that pressure-overload hypertrophy induced by thoracic aortic banding in mice resulted in an increase in L-type Ca2+ channel density but no significant alteration in Ito. Cyclosporin A prevented the hypertrophy and increase in Ca2+ current in aortic-banded mice, indicating a central role for calcineurin, a Ca2+/calmodulin–activated serine/threonine protein phosphatase.
The mechanism by which calcineurin mediates cardiac hypertrophy in mice was first demonstrated in 1999.8,9⇓ NFAT3 (nuclear factors in activated T cells) is normally heavily phosphorylated when in the cytoplasm. When NFAT3 is dephosphorylated by calcineurin, it translocates into the nucleus where it can interact with GATA4 to activate transcription of hypertrophic response genes. The importance of the calcineurin pathway in pressure-overload hypertrophy has also been demonstrated using transgenic mice overexpressing a dominant-negative mutant of calcineurin.10 It is important to note that other signaling pathways such as the protein kinase C pathway or the mitogen-activated protein kinase (MAPK) cascade have also been shown to mediate the hypertrophic response in the heart.11,12⇓
Taken together, the majority of published evidence suggests that activation of the calcineurin signaling pathway, but not a reduction in Ito density, is causally related to cardiac hypertrophy. Just when it seemed the matter was settled, Kassiri et al13 report in this issue of Circulation Research that reduction of Itof in cultured neonatal rat ventricular myocytes causes hypertrophy via a calcineurin-dependent pathway. Two approaches were used. First, Itof was blocked by heteropodatoxin, a spider toxin that specifically blocks Kv4.2 channels without any effect on Kv1.4 channels.14 Treatment with toxin for 30 to 70 hours of spontaneously contracting cultured myocytes reduced Itof by ≈50%, increased cell capacitance (an electrical measure of cell size) by 30%, and increased protein synthesis (measured by 3H-leucine incorporation) by 23%. For unknown reasons, reduction of Itof by the spider toxin also reduced the expression of Kv4.2 channels. The second approach used adenoviral-mediated infection to overexpress dominant-negative Kv4.2 channel subunits, either the N-truncated Kv4.2 subunit previously used by Backx’s group,1 or the Kv4.2W362F mutant subunit previously used in the transgenic mouse studies by Barry et al.2 Ito was reduced by ≈50% in myocytes infected with either transgene, and biophysical analyses revealed that Itof was selectively abolished. The reduction in Itof caused more than a doubling of APD90, a 47% increase in cell capacitance, and a 38% increase in 3H-leucine incorporation, changes that were suppressed by simultaneous overexpression of wild-type Kv4.2 channel subunits. In contrast, elimination of Kv1.4 channel current by overexpression of a dominant-negative transgene did not alter these measures of cell size or growth. This was an expected finding given the slow rate of Itos recovery from inactivation.
Overexpression of the dominant-negative Kv4.2 subunits also caused about a 70% increase in NFAT activity and a similar increase in calcineurin phosphatase activity.13 Coinfection with a specific noncompetitive inhibitor of calcineurin prevented the increase in cell capacitance and 3H-leucine uptake but did not prevent the decrease in Itof. The authors concluded that prolongation of APD caused by suppression of Itof increases [Ca2+]i, which activates calcineurin and subsequent transcription of hypertrophic response genes (Figure, panel B). In support of this hypothesis, prevention of spontaneous contractions and the associated increase in [Ca2+]i by treatment of cells with the L-type Ca2+ channel blocker verapamil or depolarization of cells with high [K+]o eliminated calcineurin activation and the measures of cell growth but did not prevent the reduction of Itof or prolongation of APD in electrically paced myocytes.
There is clearly a need to understand why some rodent models show an association between prolonged action potentials and hypertrophy or heart failure and others do not. Equally important is the question of whether studies in rodents provide significant insight into human disorders of cardiac repolarization. For example, despite the presence of markedly prolonged action potentials in human long-QT syndrome, there is no evidence of hypertrophy.15
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
- ↵Wickenden AD, Lee P, Sah R, Huang Q, Fishman GI, Backx PH. Targeted expression of a dominant-negative Kv4.2 K+ channel subunit in the mouse heart. Circ Res. 1999; 85: 1067–1076.
- ↵Barry DM, Xu H, Schuessler RB, Nerbonne JM. Functional knockout of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4 α subunit. Circ Res. 1998; 83: 560–567.
- ↵Guo W, Li H, London B, Nerbonne JM. Functional consequences of elimination of Ito,f and Ito,s: early afterdepolarizations, atrioventricular block, and ventricular arrhythmias in mice lacking Kv1.4 and expressing a dominant-negative Kv4 α subunit. Circ Res. 2000; 87: 73–79.
- ↵Kuo H-C, Cheng C-F, Clark RB, Lin JJ-C, Lin JL-C, Hoshijima M, Nguyen-Tran VTB, Gu Y, Ikeda Y, Chu P-H, Ross J, Giles WR, Chien KR. A defect in the channel-interacting protein 2 (KChIP2) gene leads to a complete loss of the Ito transient outward potassium current and confers genetic susceptibility to ventricular tachycardia. Cell. 2001; 107: 1–20.
- ↵Wang Z, Kutschke W, Richardson KE, Karimi M, Hill JA. Electrical remodeling in pressure-overload cardiac hypertrophy: role of calcineurin. Circulation. 2001; 104: 1657–1663.
- ↵Sussman MA, Lim HW, Gude N, Taigen T, Olson EN, Robbins J, Colbert MC, Gualberto A, Wieczorek DF, Molkentin JD. Prevention of cardiac hypertrophy in mice by calcineurin inhibition. Science. 1998; 281: 1690–1693.
- ↵Zou Y, Hiroi Y, Uozumi H, Takimoto E, Toko H, Zhu W, Kudoh S, Mizukami M, Shimoyama M, Shibasaki F, Nagai R, Yazaki Y, Komuro I. Calcineurin plays a critical role in the development of pressure overload-induced cardiac hypertrophy. Circulation. 2001; 104: 97–101.
- ↵Sugden PH. Signaling in myocardial hypertrophy: life after calcineurin? Circ Res. 1999; 84: 633–646.
- ↵Olson EN, Molkentin JD. Prevention of cardiac hypertrophy by calcineurin inhibition: hope or hype? Circ Res. 1999; 84: 623–632.
- ↵Kassiri Z, Zobel C, Nguyen T-TT, Molkentin JD, Backx PH. Reduction of Ito causes hypertrophy in neonatal rat ventricular myocytes. Circ Res. 2002; 90: 578–585.
- ↵Sanguinetti MC, Johnson JH, Hammerland LG, Kelbaygh PR, Volkmann RA, Saccomano NA, Mueller AL. Heteropodatoxins: peptides isolated from spider venom that block Kv4.2 potassium channels. Mol Pharmacol. 1997; 51: 491–498.
- ↵Nador F, Beria G, De Ferrari GM, Stramba-Badiale M, Locati EH, Lotto A, Schwartz PJ. Unsuspected echocardiographic abnormality in the long QT syndrome: diagnostic, prognostic, and pathogenetic implications. Circulation. 1991; 84: 1530–1542.