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(Circulation Research. 2005;97:1342.)
© 2005 American Heart Association, Inc.

Integrative Physiology

Targeted Deletion of Kv4.2 Eliminates I_to,f and Results in Electrical and Molecular Remodeling, With No Evidence of Ventricular Hypertrophy or Myocardial Dysfunction

Weinong Guo^*, W. Edward Jung^*, Céline Marionneau^*, Franck Aimond, Haodong Xu, Kathryn A. Yamada, Thomas L. Schwarz, Sophie Demolombe, Jeanne M. Nerbonne

From the Departments of Molecular Biology and Pharmacology (W.G., F.A., H.X., J.M.N.) and Internal Medicine (K.A.Y.), Washington University Medical School, St Louis, Mo; Department of Molecular and Cellular Physiology (W.E.J.), Stanford University Medical School, Palo Alto, Calif; Institut du Thorax (C.M., S.D.), INSERM, Nantes, France; and Program in Neurobiology (T.L.S.), Children’s Hospital, Harvard Medical School, Boston, Mass.

Correspondence to Jeanne M. Nerbonne, Department of Molecular Biology and Pharmacology, Washington University Medical School, 660 South Euclid Ave, St Louis, MO 63110. E-mail jnerbonne{at}wustl.edu

	Abstract

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Previous studies have demonstrated a role for voltage-gated K⁺ (Kv) channel subunits of the Kv4 subfamily in the generation of rapidly inactivating/recovering cardiac transient outward K⁺ 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 Kvß1, 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 Kv4.2 (Kv4.2^–/–) were examined. Unexpectedly, voltage-clamp recordings from Kv4.2^–/– ventricular myocytes revealed that I_to,f is eliminated. In addition, the slow transient outward K⁺ current, I_to,s, and the Kv1.4 protein (which encodes I_to,s) are upregulated in Kv4.2^–/– ventricles. Although Kv4.3 mRNA/protein expression is not measurably affected, KChIP2 expression is markedly reduced in Kv4.2^–/– ventricles. Similar to Kv4.3, expression of Kvß1, as well as Kv1.5 and Kv2.1, is similar in wild-type and Kv4.2^–/– ventricles. In addition, and in marked contrast to previous findings in mice expressing a truncated Kv4.2 transgene, the elimination I_to,f in Kv4.2^–/– mice does not result in ventricular hypertrophy. Taken together, these findings demonstrate not only an essential role for Kv4.2 in the generation of mouse ventricular I_to,f channels but also that the loss of I_to,f per se does not have overt pathophysiological consequences.

Key Words: I_to,s • Kv channels • remodeling • arrhythmia • hypertrophy

	Introduction

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Voltage-gated K⁺ (Kv) channels control myocardial action potential waveforms and, in most cells, multiple Kv channel types are coexpressed.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 (ß) 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 protein¹⁷ 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.^4,7 The Kv4.2DN mice display modest QT prolongation but are otherwise indistinguishable from nontransgenic littermates.^4,7 In contrast, in myocytes from mice expressing a truncated Kv4.2 subunit, Kv4.2N, that also functions as a dominant negative, I_to,f, although attenuated, is not eliminated.¹⁹ The expression of Kv4.2N was associated with cardiac hypertrophy,¹⁹ which was blocked by inhibition of calcineurin and Ca²⁺ influx.²⁰ Suppression of I_to,f in neonatal rat ventricular myocytes in vitro also reportedly results in cellular hypertrophy,²¹ and overexpression of Kv4.2 (and upregulation of I_to,f) prevents this effect.²² Although hypertrophy was attributed to the attenuation of I_to,f in these studies,^19–22 hypertrophy is not evident in Kv4.2DN⁷ or in KChIP2^–/–14 ventricles in which I_to,f is undetectable.^4,7,14

The experiments here were undertaken to explore the role of Kv4.2 in the generation of mouse ventricular I_to,f channels. Voltage-clamp recordings from myocytes isolated from mice bearing a targeted disruption of the KCND2 locus (Kv4.2^–/–) reveal that I_to,f is eliminated. Although there is no detectable effect on Kv4.3 expression, KChIP2 expression is markedly reduced in Kv4.2^–/– ventricles. The results demonstrate that the Kv4.2 subunit is required for functional cell surface expression of mouse ventricular I_to,f channels and that the loss of I_to,f does not result in hypertrophy or compromised cardiac functioning.

	Materials and Methods

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Animals 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^–/– mice and the methods/protocols used in the present study are detailed in the online data supplement available at http://circres.ahajournals.org.

	Results

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Targeted Disruption of the KCND2 (Kv4.2) Locus
In the targeting construct used to generate Kv4.2^–/– mice (Figure 1A), described in the expanded Materials and Methods section in the online data supplement, all of exon 1 was replaced with a neomycin cassette, eliminating the KCND2 coding sequence from the beginning of the N terminus to the middle of the S5–S6 (pore) loop. This approach removed essentially all of the Kv4.2 protein, thereby preventing the generation of mutant/truncated Kv4.2 protein that might, if expressed, exert dominant-negative effects on channels encoded by other Kv4 subfamily members.

View larger version (57K): [in this window] [in a new window]   Figure 1. KCND2 targeting construct and the generation of Kv4.2–/– mice. A, Schematics illustrating the membrane topology of the Kv4.2 protein and physical maps of mouse KCND2, the targeting construct and the targeted allele. The regions selected for the generation of 5' and 3' probes for Southern blot analysis are also illustrated. B, BamH1-digested genomic DNA from transfected ES cells that survived the selection procedure were screened using the 5' and 3' probes. C, Southern blot of genomic tail DNA from WT (Kv4.2+/+), homozygous targeted deletion (Kv4.2–/–), and heterozygous (Kv4.2+/–) animals probed with the 5' probe. D, Northern blot analysis of brain, heart, and liver mRNA samples from Kv4.2+/+, Kv4.2+/–, and Kv4.2–/– animals confirmed the elimination of the Kv4.2 message in Kv4.2–/– brains/hearts. E, Western blots of RV and LV membrane proteins probed with a monoclonal anti-Kv4.2 antibody confirmed that no Kv4.2 protein (arrow) is detected in Kv4.2–/– hearts.

For screening embryonic stem (ES) cells and mice, 5' and 3' probes were designed (Figure 1A) to distinguish between the targeted and endogenous KCND2 sequences. Southern blot analysis of BamH1-digested genomic DNA from ES cells that survived the selection procedure (Supplemental Materials and Methods) confirmed the presence of the targeted allele (Figure 1B). The wild-type (WT) bands are at &18 kb, and the bands corresponding to the targeted allele are at 6.2 kb and 11.8 kb for the 5' and the 3' probes (Figure 1B), respectively.

Generation and Characterization of KCND2 Targeted Deletion (Kv4.2^–/–) Mice
Blastocyst injections yielded 8 chimeric mice, 3 of which provided germline transmission on crossing with WT (FVB) mice. Congenic FVB (Kv4.2^+/–) mice were bred to establish the Kv4.2^–/– line used here. A representative Southern blot of tail DNA from the offspring of Kv4.2^+/– animals is presented in Figure 1C. Northern blot analysis of brain, heart, and liver mRNA samples from 3 of these mice reveals the presence of Kv4.2 mRNA in the brains and hearts of Kv4.2^+/+ and Kv4.2^+/– animals, whereas no Kv4.2 message is detected in Kv4.2^–/– brains/hearts (Figure 1D). Western blots confirmed robust expression of Kv4.2 protein in WT ventricles, whereas Kv4.2 is undetectable in Kv4.2^–/– ventricles (Figure 1E).

The Kv4.2^–/– mice are viable and fertile and, on gross examination, are indistinguishable from WT mice. Mean±SEM body weights in 10-week male WT and Kv4.2^–/– animals, for example, were 26.7±2.7 g (n=12) and 26.9±3.6 g (n=12), respectively. In addition, mean±SEM WT (105±4 mg) and Kv4.2^–/– (102±3 mg) heart weights are similar. Heart to body weight ratios are also not significantly different, and gross histological examination of Kv4.2^–/– hearts revealed no detectable differences from WT hearts. Similar results were obtained in animals/hearts examined at 5 to 6 months. There was also no evidence of cellular, tissue, or whole animal pathology in older Kv4.2^–/– animals.

Echocardiographic analysis of (10-week) Kv4.2^–/– hearts revealed no evidence of left ventricular (LV) hypertrophy, chamber dilation, or contractile dysfunction. Similar results were obtained in 20- to 22-week-old animals. In addition, cardiac catheterization revealed no detectable differences in hemodynamic functioning in WT and Kv4.2^–/– animals examined at 6 to 7, 9 to 10, or 21 to 22 weeks (supplemental Table I). By all available criteria, therefore, there do not appear to be any measurable pathophysiological consequences of the targeted deletion of Kv4.2.

Kv Currents Are Altered in Kv4.2^–/– Ventricular Myocytes
Peak Kv current (I_peak) densities are significantly (P<0.001) lower in myocytes isolated from the LV apex (LVA) of Kv4.2^–/– (Figure 2B), compared with WT (Figure 2A), animals (Table 1); I_K1 densities in WT and Kv4.2^–/– cells are indistinguishable (not illustrated). In addition, the waveforms of the currents in WT and Kv4.2^–/– LVA myocytes are distinct (Figure 2). In particular, the rapid component of current decay, which reflects I_to,f^2,3,7 and is prominent in WT LVA cells (Figure 2A), is not evident in Kv4.2^–/– LVA cells (Figure 2B). Kinetic analysis of the decay phases of the currents indeed revealed that the rapidly inactivating current is eliminated in all Kv4.2^–/– LVA myocytes (Table 1).

View larger version (33K): [in this window] [in a new window]   Figure 2. The rapidly inactivating/recovering Kv current is eliminated in Kv4.2–/– ventricular myocytes. A and B, Whole-cell Kv currents were recorded from WT (A) and Kv4.2–/– (B) LVA and interventricular septum myocytes in response to 4.5-sec depolarizing voltage steps to –40 to +50 mV from a holding potential of –70 mV. The currents were normalized for differences in cell size, and current densities are plotted. The rapid component of current decay, which is prominent in WT LVA cells (A), is absent in Kv4.2–/– myocytes (B). In addition, the waveforms of the currents in Kv4.2–/– LVA and septum cells are similar. C through E, To examine the kinetics of Kv current recovery from inactivation, a 3-pulse protocol was used. After inactivating the currents during 10-sec prepulses to +50 mV, cells were hyperpolarized to –70 mV for varying times (10 ms to 10 sec) before test depolarizations to +50 mV. Representative current waveforms recorded from WT (C) and Kv4.2–/– (D) LVA cells are illustrated. Current amplitudes +50 mV following each recovery period were measured and normalized to the amplitude recorded (from the same cell) following the 10-sec recovery period. E, Mean±SEM normalized peak Kv currents are plotted as a function of recovery time. The rapid component of recovery (rec&40 ms), seen in WT LVA myocytes (n=14), is not evident in Kv4.2–/– LVA cells (n=16). Peak Kv current recovery is slow (rec&1 sec) in Kv4.2–/– LVA cells.

View this table: [in this window] [in a new window]   Table 1. Kv Current Densities in WT and Kv4.2–/– Ventricular Myocytes

Previous studies have demonstrated that peak Kv current densities are lower and that I_to,f densities are variable in interventricular septum myocytes.^2,3,7 In &75% of WT septum cells, I_to,f is expressed, whereas in the other &25%, I_to,f is undetectable.² All septum cells, however, express a rapidly activating and slowly inactivating/recovering transient Kv current, I_to,s, that is not expressed in LVA myocytes.^2,3,7 In all Kv4.2^–/– septum cells, I_to,f is undetectable (Figure 2B). Peak Kv current densities are also reduced in right ventricular (RV) myocytes from Kv4.2^–/– animals (supplemental Figure I), and similar to Kv4.2^–/– LVA and septum cells, I_to,f is undetectable in Kv4.2^–/– RV myocytes (Table 1). Indeed, the Kv currents in Kv4.2^–/– RV (LVA and septum) cells are indistinguishable from those recorded from myocytes expressing the dominant-negative Kv4.2DN construct (supplemental Figure I), in which I_to,f is eliminated.^4–6

Slowly Inactivating Current Is Evident in Kv4.2^–/– LVA Myocytes
As reported previously,^2,3,7 the decay phases of the peak outward currents in WT LVA cells are well described by the sum of 2 exponentials with mean±SEM (n=27) decay time constants (_decay) of 72±4 ms and 956±45 ms. In Kv4.2^–/– LVA cells, current decay is also biexponential, although the mean±SEM (n=26) _decay were 212±19 and 1140±85 ms. These results suggest that, in the absence of I_to,f, a slowly inactivating current component, that is not evident in WT LVA cells, is expressed. The _decay of this current is similar to the _decay for I_to,s in WT septum cells, suggesting that elimination of I_to,f in LVA myocytes results in I_to,s upregulation. Similar results were obtained on analysis of the Kv currents in Kv4.2^–/– RV cells (supplemental Figure I).

Another interpretation of the appearance of the slowly decaying current in Kv4.2^–/– LVA and RV cells might be that this current reflects the expression of homotetrameric Kv4.3 channels. Indeed, it is well documented that Kv4.3 can form functional homomeric channels in heterologous expression systems.¹⁰ In addition, in large mammals, including humans, Kv4.2 is not expressed and functional I_to,f channels appear to be encoded by Kv4.3.^1,11,12 Subsequent experiments, therefore, were aimed at distinguishing between these 2 possibilities.

No Fast Component of Recovery Is Evident in Kv4.2^–/– Ventricular Myocytes
In addition to rapid inactivation, mouse ventricular I_to,f is characterized by rapid recovery from inactivation.^2,3,7 Homotetrameric Kv4.2 or Kv4.3 channels also recover from inactivation rapidly, although the time constants of recovery (_rec) for homomeric Kv4.2 or Kv4.3 channels in myocytes or in heterologous cells are twice those of heteromeric Kv4.2/Kv4.3 channels.¹⁰ Mouse ventricular I_to,s channels, in contrast, recover slowly from inactivation with time constants on the order of 1 sec.^1,10 Subsequent experiments, therefore, were focused on examining recovery kinetics in WT and Kv4.2^–/– myocytes (Figure 2C and 2D). As previously reported,^2,3,7 recovery of I_to,f in WT LVA myocytes follows a monoexponential time course with a mean±SEM _rec of 37±6 ms (Figure 2E). Recovery in Kv4.2^–/– LVA cells is also best described by a single exponential, but with a mean±SEM _rec of 1075±125 ms (Figure 2E). There is no fast component of recovery in Kv4.2^–/– LVA cells (Figure 2E), suggesting that the slowly inactivating Kv current in these cells does not reflect the expression of Kv4.3 homotetrameric channels. In addition, the _rec (& 1 sec) in Kv4.2^–/– LVA myocytes is similar to the _rec for I_to,s in WT septum cells.^2,5,7 Similar results were obtained in experiments on Kv4.2^–/– RV cells.

No Heteropodatoxin-Sensitive Currents Are Evident in Kv4.2^–/– Myocytes
The results presented above suggest that the "novel" slowly inactivating/recovering Kv current evident in Kv4.2^–/– LVA (and RV) myocytes likely does not reflect the presence of homomeric Kv4.3 channels. To further explore this hypothesis, pharmacological experiments were completed. It has been previously demonstrated that mouse ventricular I_to,f is selectively reduced by nanomolar concentrations of heteropodatoxin (HpTx)-2 or -3,^2,23 toxins that are selective for Kv4 channels.²⁴ Subsequent experiments, therefore, were focused on determining whether there were residual HpTx-sensitive currents in Kv4.2^–/– myocytes. In WT LVA and septum cells, 300 nmol/L HpTx-3 blocks a substantial component of the rapidly inactivating current (Figure 3A). The waveforms of the HpTx-3 sensitive currents are consistent with the selective attenuation of I_to,f. In contrast, Kv currents in Kv4.2^–/– LVA and septum cells are not measurably affected by 300 nmol/L HpTx-3 (Figure 3B). The simplest interpretation of these results is that there are no Kv4-encoded currents in Kv4.2^–/– cells, ie, that Kv4.3 does not form functional homotetrameric channels in mouse ventricular myocytes in the absence of Kv4.2. Taken together, these results also indicate that the slowly inactivating/recovering current identified in Kv4.2^–/– LVA (and RV) myocytes reflects the presence of I_to,s or some other, previously undescribed, Kv current.

View larger version (20K): [in this window] [in a new window]   Figure 3. No HpTx-sensitive Kv currents are evident in Kv4.2–/– ventricular myocytes. Kv currents were recorded from WT (A) and Kv4.2–/– (B) LVA and septum myocytes as described in the legend to Figure 2 before, during, and after the application of 300 nmol/L HpTx-3. In the upper records of A and B, the currents recorded in the absence (black) and presence (red) of 300 nmol/L HpTx-3 are superimposed. The HpTx-3–sensitive currents were obtained by digital offline subtraction of the records in the absence and in the presence of the toxin. No HpTx-3–sensitive currents are detected in Kv4.2–/– LVA or septum cells.

Functional Consequences of the Targeted Deletion of Kv4.2
To examine the functional consequences of the deletion of KCND2, telemetric electrocardiographic (ECG) recordings were obtained from adult (10-week) WT and Kv4.2^–/– animals. As evident in the representative recordings in Figure 4A, the morphologies of the QRS complexes and P waves in WT and Kv4.2^–/– animals are indistinguishable. In addition, there were no significant differences in the durations of the QT, PR, QRS, or RR intervals in WT and Kv4.2^–/– mice (Table 2). Mean heart rates measured in WT and Kv4.2^–/– animals are also indistinguishable, and mean corrected QT (QT_c) intervals in 10-week Kv4.2^–/– and WT animals are, therefore, not significantly different (Table 2). Similar results were obtained in animals monitored at 5 to 6 months of age (Table 2). Analysis of the ECG records obtained from Kv4.2^–/– animals during 48 hours of continuous monitoring also revealed no evidence of rhythm disturbances (not illustrated).

View larger version (24K): [in this window] [in a new window]   Figure 4. ECG and ventricular action potential waveforms in WT and Kv4.2–/– mice are indistinguishable. A, No significant differences in heart rates and/or in the morphologies of the P waves, QRS complexes, or QT intervals are evident in telemetric ECG recordings from WT and Kv4.2–/– animals. B, Action potential waveforms in WT and Kv4.2–/– LVA cells are indistinguishable. C, There are no significant differences in mean±SEM action potential durations (APD) at 50%, 75%, or 90% repolarization in WT and Kv4.2–/– LVA myocytes.

View this table: [in this window] [in a new window]   Table 2. ECG Parameters Are Unaffected by the Targeted Deletion of Kv4.2

The similarities in QT (QT_c) intervals in Kv4.2^–/– and WT animals suggest that ventricular (action potential) repolarization is not affected by the deletion of Kv4.2, in spite of the loss of I_to,f. Consistent with this hypothesis, action potential waveforms in Kv4.2^–/– and WT LVA myocytes are indistinguishable (Figure 4B), and mean±SEM action potential durations at 50% (APD₅₀), 75% (APD₇₅), and 90% (APD₉₀) repolarization in WT and Kv4.2^–/– LVA myocytes are not significantly different (Figure 4C).

Molecular Basis of Kv Channel Remodeling in Kv4.2^–/– Ventricles
The electrophysiological findings that I_to,f is eliminated and that there is no evidence for the presence of Kv4.3 channels in Kv4.2^–/– ventricular myocytes were unexpected in light of previous studies demonstrating that Kv4.3 forms functional channels in heterologous systems^10,12,16 and that I_to,f in large mammals reflects homotetrameric Kv4.3 channels.^11,12 These results suggested the interesting possibility that Kv4.3 expression might be affected by the deletion of Kv4.2. In addition, the electrophysiological studies demonstrated the functional upregulation of a slowly inactivating/recovering Kv current in LVA and RV myocytes with properties similar to Kv1.4-encoded I_to,s channels in WT septum cells, suggesting that Kv1.4 expression might also be affected in Kv4.2^–/– ventricles.

Subsequent experiments were focused on exploring the hypothesis that molecular remodeling occurs in Kv4.2^–/– ventricles. In initial experiments, the expression levels of the transcripts encoding Kv subunits and a variety of other ion channel subunits (supplemental Table II) were examined using TaqMan Low Density microarrays²⁵ (Supplemental Materials and Methods). These analyses revealed little evidence of molecular remodeling at the transcript level. The expression levels of the various subunits shown previously to contribute to I_to,f, KCND3 (Kv4.3),¹⁰ KCNIP2 (KChIP2),¹⁰ and KCNAB1 (Kvß1)²³ are not measurably different in Kv4.2^–/– and WT ventricles (Figure 5A). In Kv4.2^–/– ventricles, there is a small, but statistically significant (P<0.01) increase in KCNA5 (Kv1.5), which encodes 1 component of I_K,slow,^8,9 whereas the expression levels of several other Kv channel subunits (Figure 5A), as well as of a host of other channel genes (supplemental Figure II), are not significantly different in WT and Kv4.2^–/– ventricles.

View larger version (13K): [in this window] [in a new window]   Figure 5. Molecular remodeling in Kv4.2–/– ventricles. The expression levels of ion channel subunit and regulatory genes (supplemental Table II) in WT (n=5) and Kv4.2–/– (n=5) 10-week ventricles were examined using TaqMan-based Low Density Arrays25 (supplemental Materials and Methods); values are means±SEM. KCNA5 (Kv1.5) expression is increased significantly (P<0.01) in Kv4.2–/– ventricles, whereas the expression of other Kv and ß subunit transcripts, including KCND3 (Kv4.3) and KChIP2 (KChiP2), is similar in WT and Kv4.2–/– ventricles. B, Mean±SEM changes in Kv subunit expression in 10-week Kv4.2–/– (n=10) and WT (n=10) ventricles were also examined using SYBR green quantitative RT-PCR; primers are provided in supplemental Table III. These analyses confirmed the increase in KCNA5 and, in addition, revealed that KCNIP2 expression is decreased (by &20%) significantly (P<0.01) in Kv4.2–/–, compared with WT, ventricles. The difference in KCNIP2 expression here may reflect the fact that the primers used detect the KCNIP2a, -b, and -c variants, whereas the TaqMan analysis only detects the KCNIP2a and -b variants. C, The decrease in KCNIP2 expression is also evident in 5-month Kv4.2–/– ventricles (n=6).

To validate the TaqMan low-density array data, SYBR green quantitative RT-PCR was performed on Kv4.2^–/– and WT ventricles using Kv subunit specific primers (supplemental Table III). These analyses also revealed no significant differences in Kv1.4, Kv4.3, Kvß1, or Kv2.1 expression in Kv4.2^–/– and WT ventricles (Figure 5B) and a small, but statistically significant (P<0.05), increase in Kv1.5 (Figure 5B). Using primers that detect the KCNIP2a, -b, and -c splice variants, the SYBR green RT-PCR revealed a small (&20%), but statistically significant (P<0.01), reduction in KChIP2 expression in Kv4.2^–/– ventricles (Figure 5B). The small reduction in KCNIP2 expression is also evident in samples from 5-month-old Kv4.2^–/– animals (Figure 5F). Taken together, these combined analyses (Figure 5 and supplemental Figure II) demonstrate very little molecular remodeling at the transcript level in Kv4.2^–/– ventricles.

To determine the effects of the targeted deletion of Kv4.2 on the expression levels of Kv subunit proteins, Western blot analyses of LV and RV membrane proteins were completed. As illustrated in Figure 6, Kv4.3 expression is similar in WT and Kv4.2^–/– LV and RV samples. Expression of Kvß1, Kv1.5, and Kv2.1 is also unaffected by the elimination of Kv4.2 (Figure 6). In contrast, KChIP2 protein expression is markedly (P<0.001) reduced in Kv4.2^–/– (right and left) ventricles (Figure 6). Although the quantitative RT-PCR analysis did reveal a (small) reduction in KCNIP2 expression, the reduction in KChIP2 protein (&90%) is substantially greater than would be expected by the reduction (&20%) in message expression alone.

View larger version (34K): [in this window] [in a new window]   Figure 6. KChIP2 protein expression is markedly reduced in Kv4.2–/– ventricles. A, Western blots of fractionated proteins from adult (10-week) WT and Kv4.2–/– RV and LV, probed with anti-Kv4.3, anti-KChIP2, anti-Kvß1, and anti-Kv1.5 antibodies. B, Films from individual experiments were scanned, and the densities of the Kv4.3, KChIP2, Kvß1, Kv1.4, Kv1.5, and Kv2.1 bands in the Kv4.2–/– (n=8) and WT (n=8) samples were measured. The densities of WT LV and Kv4.2–/– RV and LV samples were normalized to the WT RV sample on the same blot; mean±SEM densities are plotted in B. Kv1.4 protein is increased modestly (by &20%; P<0.05), whereas KChIP2 is reduced markedly (P0.001) in Kv4.2–/– (n=8), compared with WT (n=8), RV and LV. There are no significant differences in Kv4.3, Kvß1, Kv1.5, or Kv2.1 expression.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Targeted Disruption of Kv4.2 Eliminates Mouse Ventricular I_to,f
The results presented here demonstrate that I_to,f is eliminated in myocytes isolated from the left and right ventricles and from the interventricular septum of Kv4.2^–/– mice. In addition, there are no rapidly inactivating/recovering Kv currents and no residual HpTX-3–sensitive currents detected in Kv4.2^–/– ventricular myocytes. There is no evidence, therefore, that Kv4.3 forms functional homomeric Kv channels in Kv4.2^–/– mouse ventricles. The elimination of I_to,f is accompanied, however, by an increase in a slowly inactivating/recovering Kv current similar to I_to,s in WT septum cells. These observations, ie, the loss of I_to,f and the upregulation of I_to,s, are similar to previous findings in mice expressing a mutant Kv4.2 subunit, Kv4.2DN, that functions as a dominant negative.^4,7 In contrast to cells/animals expressing Kv4.2DN,^4,7 however, the loss of I_to,f in Kv4.2^–/– hearts is not reflected in altered action potential waveforms or electrocardiograms.

The fact that action potential waveforms and QT_c intervals in Kv4.2^–/– and WT myocytes/animals are indistinguishable suggests that the upregulation of I_to,s compensates for the loss of I_to,f. Action potential and QT_c prolongation, however, are evident in Kv4.2DN mice,^4,7 in which I_to,f is also eliminated and I_to,s is also upregulated. There is a statistically significant (P<0.001) difference in the magnitude of I_to,s measured in Kv4.2^–/– RV cells compared with Kv4.2DN RV cells⁷ that may account, at least in part, for the phenotypic differences in the Kv4.2^–/– and Kv4.2DN animals. In RV cells from Kv4.2^–/– and Kv4.2DN animals, mean±SEM I_to,s densities were 15.6±1.9 pA/pF and 7.5±1.4 pA/pF, respectively. The densities of I_to,s in Kv4.2^–/– and Kv4.2DN LVA myocytes, however, are similar, suggesting that the upregulation of I_to,s alone does not explain the normalization of action potentials in Kv4.2^–/– LVA myocytes. It seems likely that more complex electrical and molecular remodeling occurs as a result of the elimination of Kv4.2 and the loss of I_to,f, ie, remodeling that does not occur in Kv4.2DN ventricles. Further experiments aimed at testing this hypothesis and determining the molecular mechanisms controlling remodeling in these 2 models are clearly warranted.

Molecular Remodeling in Kv4.2^–/– Ventricles
It has been suggested that electrical remodeling (I_to,s upregulation) in Kv4.2DN ventricles likely reflects transcriptional regulation of KCNA4 (Kv1.4).²⁶ Although the experiments here revealed no differences in Kv1.4 message, there is a small, but statistically significant (P<0.05), increase in Kv1.4 protein expression in Kv4.2^–/– ventricles. It is unclear whether the increase in Kv1.4 protein alone underlies the upregulation of I_to,s in Kv4.2^–/– ventricles or, alternatively, whether there are additional factors, such as interactions with auxiliary subunits^3,27 or changes in the mechanisms regulating channel trafficking and cell surface expression.^1,28–30

The finding that Kv4.3 message/protein expression is not measurably affected in Kv4.2^–/– ventricles lacking I_to,f clearly suggests that mouse Kv4.3 subunits cannot coassemble and/or traffic properly in the absence of Kv4.2. Subunit-specific differences in the regulation of channel assembly and trafficking have been described for Kir subunits.^31,32 It is certainly also possible that assembled homomeric Kv4.3 channels do not traffic properly to the cell surface, owing to changes in the properties and/or expression of additional factors, such as auxiliary subunits²⁷ or cytoskeletal components.^28–30 In this context, it is interesting that KChIP2 protein expression is markedly reduced in Kv4.2^–/– ventricles. There are also modest changes in KCNIP2 expression, although it seems almost certain that the predominant effect in Kv4.2^–/– ventricles is posttranscriptional, reflecting changes in the synthesis or the degradation of the KChIP2 protein.

Essential Role for Kv4.2 in the Generation of Mouse Ventricular I_to,f Channels
Previous studies have demonstrated that Kv4.2 and Kv4.3 coimmunoprecipitate from adult mouse ventricles, an observation interpreted as suggesting that Kv4.2 and Kv4.3 associate to form heteromeric I_to,f channels.¹⁰ In addition, Kv4.2 is differentially expressed in mouse ventricles and differences in Kv4.2 expression parallel regional variations in I_to,f densities.¹⁰ The observation here that there are no rapidly inactivating/recovering Kv currents, as well as no HpTx-sensitive Kv currents, in Kv4.2^–/– ventricles suggests that, in the absence of Kv4.2, Kv4.3 cannot form functional cell surface Kv channels in mouse ventricles. Kv4.2, therefore, plays an essential role in the generation of functional cell surface mouse ventricular I_to,f channels. These findings were unexpected in light of previous studies suggesting that Kv4.3 forms functional channels in heterologous systems^10,12,27 and that homomeric Kv4.3 channels underlie I_to,f in large mammals, including humans.^11,12 The absence of functional I_to,f channels and the loss of the KChIP2 protein in Kv4.2^–/– ventricles suggest that additional subunits may be involved in regulating Kv4.3-encoded I_to,f channel assembly and/or trafficking in human heart.

Relationship With Previous Studies
In ventricular myocytes isolated from Kv4.2DN mice, I_to,f is selectively eliminated and action potentials and QT intervals are prolonged.^4,7 In contrast, I_to,f density is reduced (but not eliminated) in a subset of ventricular myocytes isolated from young (2- to 3-week-old) animals expressing a truncated Kv4.2 subunit, Kv4.2N, that also functions as a dominant negative.¹⁹ It was also reported that, as Kv4.2N animals age, hypertrophy, chamber dilation, and interstitial fibrosis are evident.¹⁹ At 10 to 12 weeks, the Kv4.2N animals develop congestive heart failure and increased incidence of sudden death.¹⁹ Recordings from myocytes isolated from older Kv4.2N animals revealed marked reductions in I_K1, as well as I_to,f, densities.¹⁹ Although the pathological effects of Kv4.2N expression were attributed to attenuation of I_to,f, this interpretation is inconsistent with the results obtained here, as well as those obtained previously in the studies on the Kv4.2DN transgenics.^4,7 There is no evidence of hypertrophy or compromised functioning in Kv4.2^–/– or Kv4.2DN hearts. Clearly, the absence of pathology in Kv4.2^–/– and Kv4.2 DN animals argues strongly against a causal relationship between the loss of I_to,f and cardiac hypertrophy, as previously suggested.^19–22

	Acknowledgments

 
Financial support provided by the American Heart Association (Postdoctoral Fellowships to F.A. and W.G.), the NIH (grants HL-034161 and HL-066388 to J.M.N.; HL-066385 to K.A.Y.; GM-042376 to T.L.S.), and the Centre National de la Recherche Scientifique is gratefully acknowledged. The studies here were performed on animals maintained in a facility supported by the National Center for Research Resources (C06 RR015502). We thank Dr Joshua Zaritsky for the phage clone used to generate the targeting vector and Dr Andras Nagy for the ES cells. We also thank Rick Wilson for expert technical assistance and Drs Huilin Li and Bin Ye for helpful discussions.

	Footnotes

 
*These authors contributed equally to this study.

Original received September 8, 2005; revision received October 19, 2005; accepted November 3, 2005.

	References

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