Targeted Deletion of Kv4.2 Eliminates Ito,f and Results in Electrical and Molecular Remodeling, With No Evidence of Ventricular Hypertrophy
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循环研究杂志 2005年第12期
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
Program in Neurobiology (T.L.S.), Children’s Hospital, Harvard Medical School, Boston, Mass.
Abstract
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, Ito,f, channels. Biochemical studies suggest that mouse ventricular Ito,f channels reflect the heteromeric assembly of Kv4.2 and Kv4.3 with the accessory subunits, KChIP2 and Kv1, and that Kv4.2 is the primary determinant of regional differences in (mouse ventricular) Ito,f densities. Interestingly, the phenotypic consequences of manipulating Ito,f expression in different mouse models are distinct. In the experiments here, the effects of the targeted deletion of Kv4.2 (Kv4.2eC/eC) were examined. Unexpectedly, voltage-clamp recordings from Kv4.2eC/eC ventricular myocytes revealed that Ito,f is eliminated. In addition, the slow transient outward K+ current, Ito,s, and the Kv1.4 protein (which encodes Ito,s) are upregulated in Kv4.2eC/eC ventricles. Although Kv4.3 mRNA/protein expression is not measurably affected, KChIP2 expression is markedly reduced in Kv4.2eC/eC ventricles. Similar to Kv4.3, expression of Kv1, as well as Kv1.5 and Kv2.1, is similar in wild-type and Kv4.2eC/eC ventricles. In addition, and in marked contrast to previous findings in mice expressing a truncated Kv4.2 transgene, the elimination Ito,f in Kv4.2eC/eC 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 Ito,f channels but also that the loss of Ito,f per se does not have overt pathophysiological consequences.
Key Words: Ito,s Kv channels remodeling arrhythmia hypertrophy
Introduction
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 () subunits in the generation of these channels.1 In adult mouse ventricles, for example, multiple Kv currents are coexpressed.2eC10 All available evidence suggests that subunits of the Kv4 subfamily underlie fast inactivating and recovering cardiac transient outward, Ito,f, channels.1 Biochemical studies suggest that mouse ventricular Ito,f channels reflect the heteromeric assembly of Kv4.2 and Kv4.3.10 In large mammals, however, Kv4.2 is not expressed, and Ito,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 Ito,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 Ito,f is eliminated in ventricular myocytes isolated from mice in which the KChIP2 locus was disrupted.14 In canine ventricles, KChIP2 message15,16 and protein17 expression parallel variations in Ito,f densities, suggesting that KChIP2 is the primary determinant of Ito,f gradients.15,17 In rodent ventricles, however, KChIP2 is uniformly expressed, and regional differences in Ito,f densities are correlated with heterogeneities in Kv4.2 expression.10,18
Interestingly, the phenotypic consequences of manipulating Ito,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 Ito,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, Ito,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 Ito,f in neonatal rat ventricular myocytes in vitro also reportedly results in cellular hypertrophy,21 and overexpression of Kv4.2 (and upregulation of Ito,f) prevents this effect.22 Although hypertrophy was attributed to the attenuation of Ito,f in these studies,19eC22 hypertrophy is not evident in Kv4.2DN7 or in KChIP2eC/eC14 ventricles in which Ito,f is undetectable.4,7,14
The experiments here were undertaken to explore the role of Kv4.2 in the generation of mouse ventricular Ito,f channels. Voltage-clamp recordings from myocytes isolated from mice bearing a targeted disruption of the KCND2 locus (Kv4.2eC/eC) reveal that Ito,f is eliminated. Although there is no detectable effect on Kv4.3 expression, KChIP2 expression is markedly reduced in Kv4.2eC/eC ventricles. The results demonstrate that the Kv4.2 subunit is required for functional cell surface expression of mouse ventricular Ito,f channels and that the loss of Ito,f does not result in hypertrophy or compromised cardiac functioning.
Materials and Methods
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 Kv4.2eC/eC 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) Locus
In the targeting construct used to generate Kv4.2eC/eC 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 S5eCS6 (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.
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 (Kv4.2eC/eC) Mice
Blastocyst injections yielded 8 chimeric mice, 3 of which provided germline transmission on crossing with WT (FVB) mice. Congenic FVB (Kv4.2+/eC) mice were bred to establish the Kv4.2eC/eC line used here. A representative Southern blot of tail DNA from the offspring of Kv4.2+/eC 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+/eC animals, whereas no Kv4.2 message is detected in Kv4.2eC/eC brains/hearts (Figure 1D). Western blots confirmed robust expression of Kv4.2 protein in WT ventricles, whereas Kv4.2 is undetectable in Kv4.2eC/eC ventricles (Figure 1E).
The Kv4.2eC/eC mice are viable and fertile and, on gross examination, are indistinguishable from WT mice. Mean±SEM body weights in 10-week male WT and Kv4.2eC/eC 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 Kv4.2eC/eC (102±3 mg) heart weights are similar. Heart to body weight ratios are also not significantly different, and gross histological examination of Kv4.2eC/eC 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 Kv4.2eC/eC animals.
Echocardiographic analysis of (10-week) Kv4.2eC/eC 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 Kv4.2eC/eC 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 Kv4.2eC/eC Ventricular Myocytes
Peak Kv current (Ipeak) densities are significantly (P<0.001) lower in myocytes isolated from the LV apex (LVA) of Kv4.2eC/eC (Figure 2B), compared with WT (Figure 2A), animals (Table 1); IK1 densities in WT and Kv4.2eC/eC cells are indistinguishable (not illustrated). In addition, the waveforms of the currents in WT and Kv4.2eC/eC LVA myocytes are distinct (Figure 2). In particular, the rapid component of current decay, which reflects Ito,f2,3,7 and is prominent in WT LVA cells (Figure 2A), is not evident in Kv4.2eC/eC LVA cells (Figure 2B). Kinetic analysis of the decay phases of the currents indeed revealed that the rapidly inactivating current is eliminated in all Kv4.2eC/eC LVA myocytes (Table 1).
Previous studies have demonstrated that peak Kv current densities are lower and that Ito,f densities are variable in interventricular septum myocytes.2,3,7 In &75% of WT septum cells, Ito,f is expressed, whereas in the other &25%, Ito,f is undetectable.2 All septum cells, however, express a rapidly activating and slowly inactivating/recovering transient Kv current, Ito,s, that is not expressed in LVA myocytes.2,3,7 In all Kv4.2eC/eC septum cells, Ito,f is undetectable (Figure 2B). Peak Kv current densities are also reduced in right ventricular (RV) myocytes from Kv4.2eC/eC animals (supplemental Figure I), and similar to Kv4.2eC/eC LVA and septum cells, Ito,f is undetectable in Kv4.2eC/eC RV myocytes (Table 1). Indeed, the Kv currents in Kv4.2eC/eC RV (LVA and septum) cells are indistinguishable from those recorded from myocytes expressing the dominant-negative Kv4.2DN construct (supplemental Figure I), in which Ito,f is eliminated.4eC6
Slowly Inactivating Current Is Evident in Kv4.2eC/eC 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 Kv4.2eC/eC 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 Ito,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 Ito,s in WT septum cells, suggesting that elimination of Ito,f in LVA myocytes results in Ito,s upregulation. Similar results were obtained on analysis of the Kv currents in Kv4.2eC/eC RV cells (supplemental Figure I).
Another interpretation of the appearance of the slowly decaying current in Kv4.2eC/eC 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 Ito,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 Kv4.2eC/eC Ventricular Myocytes
In addition to rapid inactivation, mouse ventricular Ito,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 Ito,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 Kv4.2eC/eC myocytes (Figure 2C and 2D). As previously reported,2,3,7 recovery of Ito,f in WT LVA myocytes follows a monoexponential time course with a mean±SEM rec of 37±6 ms (Figure 2E). Recovery in Kv4.2eC/eC 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 Kv4.2eC/eC 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 Kv4.2eC/eC LVA myocytes is similar to the rec for Ito,s in WT septum cells.2,5,7 Similar results were obtained in experiments on Kv4.2eC/eC RV cells.
No Heteropodatoxin-Sensitive Currents Are Evident in Kv4.2eC/eC Myocytes
The results presented above suggest that the "novel" slowly inactivating/recovering Kv current evident in Kv4.2eC/eC 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 Ito,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 Kv4.2eC/eC 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 Ito,f. In contrast, Kv currents in Kv4.2eC/eC 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 Kv4.2eC/eC 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 Kv4.2eC/eC LVA (and RV) myocytes reflects the presence of Ito,s or some other, previously undescribed, Kv current.
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 Kv4.2eC/eC animals. As evident in the representative recordings in Figure 4A, the morphologies of the QRS complexes and P waves in WT and Kv4.2eC/eC animals are indistinguishable. In addition, there were no significant differences in the durations of the QT, PR, QRS, or RR intervals in WT and Kv4.2eC/eC mice (Table 2). Mean heart rates measured in WT and Kv4.2eC/eC animals are also indistinguishable, and mean corrected QT (QTc) intervals in 10-week Kv4.2eC/eC 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 Kv4.2eC/eC animals during 48 hours of continuous monitoring also revealed no evidence of rhythm disturbances (not illustrated).
The similarities in QT (QTc) intervals in Kv4.2eC/eC and WT animals suggest that ventricular (action potential) repolarization is not affected by the deletion of Kv4.2, in spite of the loss of Ito,f. Consistent with this hypothesis, action potential waveforms in Kv4.2eC/eC 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 Kv4.2eC/eC LVA myocytes are not significantly different (Figure 4C).
Molecular Basis of Kv Channel Remodeling in Kv4.2eC/eC Ventricles
The electrophysiological findings that Ito,f is eliminated and that there is no evidence for the presence of Kv4.3 channels in Kv4.2eC/eC ventricular myocytes were unexpected in light of previous studies demonstrating that Kv4.3 forms functional channels in heterologous systems10,12,16 and that Ito,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 Ito,s channels in WT septum cells, suggesting that Kv1.4 expression might also be affected in Kv4.2eC/eC ventricles.
Subsequent experiments were focused on exploring the hypothesis that molecular remodeling occurs in Kv4.2eC/eC 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 Ito,f, KCND3 (Kv4.3),10 KCNIP2 (KChIP2),10 and KCNAB1 (Kv1)23 are not measurably different in Kv4.2eC/eC and WT ventricles (Figure 5A). In Kv4.2eC/eC ventricles, there is a small, but statistically significant (P<0.01) increase in KCNA5 (Kv1.5), which encodes 1 component of IK,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 Kv4.2eC/eC ventricles.
To validate the TaqMan low-density array data, SYBR green quantitative RT-PCR was performed on Kv4.2eC/eC and WT ventricles using Kv subunit specific primers (supplemental Table III). These analyses also revealed no significant differences in Kv1.4, Kv4.3, Kv1, or Kv2.1 expression in Kv4.2eC/eC 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 Kv4.2eC/eC ventricles (Figure 5B). The small reduction in KCNIP2 expression is also evident in samples from 5-month-old Kv4.2eC/eC animals (Figure 5F). Taken together, these combined analyses (Figure 5 and supplemental Figure II) demonstrate very little molecular remodeling at the transcript level in Kv4.2eC/eC 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 Kv4.2eC/eC LV and RV samples. Expression of Kv1, 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 Kv4.2eC/eC (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.
Discussion
Targeted Disruption of Kv4.2 Eliminates Mouse Ventricular Ito,f
The results presented here demonstrate that Ito,f is eliminated in myocytes isolated from the left and right ventricles and from the interventricular septum of Kv4.2eC/eC mice. In addition, there are no rapidly inactivating/recovering Kv currents and no residual HpTX-3eCsensitive currents detected in Kv4.2eC/eC ventricular myocytes. There is no evidence, therefore, that Kv4.3 forms functional homomeric Kv channels in Kv4.2eC/eC mouse ventricles. The elimination of Ito,f is accompanied, however, by an increase in a slowly inactivating/recovering Kv current similar to Ito,s in WT septum cells. These observations, ie, the loss of Ito,f and the upregulation of Ito,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 Ito,f in Kv4.2eC/eC hearts is not reflected in altered action potential waveforms or electrocardiograms.
The fact that action potential waveforms and QTc intervals in Kv4.2eC/eC and WT myocytes/animals are indistinguishable suggests that the upregulation of Ito,s compensates for the loss of Ito,f. Action potential and QTc prolongation, however, are evident in Kv4.2DN mice,4,7 in which Ito,f is also eliminated and Ito,s is also upregulated. There is a statistically significant (P<0.001) difference in the magnitude of Ito,s measured in Kv4.2eC/eC RV cells compared with Kv4.2DN RV cells7 that may account, at least in part, for the phenotypic differences in the Kv4.2eC/eC and Kv4.2DN animals. In RV cells from Kv4.2eC/eC and Kv4.2DN animals, mean±SEM Ito,s densities were 15.6±1.9 pA/pF and 7.5±1.4 pA/pF, respectively. The densities of Ito,s in Kv4.2eC/eC and Kv4.2DN LVA myocytes, however, are similar, suggesting that the upregulation of Ito,s alone does not explain the normalization of action potentials in Kv4.2eC/eC 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 Ito,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 Kv4.2eC/eC Ventricles
It has been suggested that electrical remodeling (Ito,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 Kv4.2eC/eC ventricles. It is unclear whether the increase in Kv1.4 protein alone underlies the upregulation of Ito,s in Kv4.2eC/eC ventricles or, alternatively, whether there are additional factors, such as interactions with auxiliary subunits3,27 or changes in the mechanisms regulating channel trafficking and cell surface expression.1,28eC30
The finding that Kv4.3 message/protein expression is not measurably affected in Kv4.2eC/eC ventricles lacking Ito,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.28eC30 In this context, it is interesting that KChIP2 protein expression is markedly reduced in Kv4.2eC/eC ventricles. There are also modest changes in KCNIP2 expression, although it seems almost certain that the predominant effect in Kv4.2eC/eC 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 Ito,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 Ito,f channels.10 In addition, Kv4.2 is differentially expressed in mouse ventricles and differences in Kv4.2 expression parallel regional variations in Ito,f densities.10 The observation here that there are no rapidly inactivating/recovering Kv currents, as well as no HpTx-sensitive Kv currents, in Kv4.2eC/eC 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 Ito,f channels. These findings were unexpected in light of previous studies suggesting that Kv4.3 forms functional channels in heterologous systems10,12,27 and that homomeric Kv4.3 channels underlie Ito,f in large mammals, including humans.11,12 The absence of functional Ito,f channels and the loss of the KChIP2 protein in Kv4.2eC/eC ventricles suggest that additional subunits may be involved in regulating Kv4.3-encoded Ito,f channel assembly and/or trafficking in human heart.
Relationship With Previous Studies
In ventricular myocytes isolated from Kv4.2DN mice, Ito,f is selectively eliminated and action potentials and QT intervals are prolonged.4,7 In contrast, Ito,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 IK1, as well as Ito,f, densities.19 Although the pathological effects of Kv4.2N expression were attributed to attenuation of Ito,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 Kv4.2eC/eC or Kv4.2DN hearts. Clearly, the absence of pathology in Kv4.2eC/eC and Kv4.2 DN animals argues strongly against a causal relationship between the loss of Ito,f and cardiac hypertrophy, as previously suggested.19eC22
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.
These authors contributed equally to this study.
References
Nerbonne JM, Kass RS. Molecular physiology of cardiac repolarization. Physiol Rev. 2005; 85: 1205eC1253.
Xu H, Guo W, Nerbonne JM. Four kinetically distinct depolarization-activated K+ currents in adult mouse ventricular myocytes. J Gen Physiol. 1999; 113: 661eC678.
Brunet S, Aimond F, Li H, Guo W, Eldstrom J, Fedida D, Yamada KA, Nerbonne JM. Heterogeneous expression of repolarizing, voltage-gated K+ currents in adult mouse ventricles. J Physiol. 2004; 559: 103eC120.
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 alpha subunit. Circ Res. 1998; 83: 560eC567.
Guo W, Xu H, London B, Nerbonne JM. Molecular basis of transient outward K+ current diversity in mouse ventricular myocytes. J Physiol. 1999; 521: 587eC599.
Xu H, Barry DM, Li H, Brunet S, Nerbonne JM. Attenuation of the slow component of delayed rectification, action potential prolongation, and triggered activity in mice expressing a dominant-negative Kv2 alpha subunit. Circ Res. 1999; 85: 623eC633.
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 alpha subunit. Circ Res. 2000; 87: 73eC79, 2000.
London B, Guo W, Pan XH, Lee JS, Shusterman V, Logothetis DA, Nerbonne JM, Hill JA. Targeted replacement of Kv1.5 in the mouse leads to loss of the 4-aminoyridine-sensitive component of IK,slow and resistance to drug induced QT prolongation. Circ Res. 2001; 88: 940eC946.
Li H, Guo W, Yamada KA, Nerbonne JM. Selective elimination of the 4-AP-sensitive component of delayed rectification, IK,slow1, in mouse ventricular myocytes expressing a dominant negative Kv1.5 alpha subunit. Am J Physiol. 2004; 286: H319eCH328.
Guo W, Li H, Aimond F, Johns DC, Rhodes KJ, Trimmer JS, Nerbonne JM. Role of heteromultimers in the generation of myocardial transient outward K+ currents. Circ Res. 2002; 90: 586eC593.
Dixon JE, Shi W, Wang HS, McDonald C, Yu H, Wymore RS, Cohen IS, McKinnon D. Role of the Kv4.3 K+ channel in ventricular muscle. A molecular correlate for the transient outward current. Circ Res. 1996; 79: 659eC668.
Kong W, Po S, Yamagishi T, Ashen MD, Stetten G, Tomaselli GF. Isolation and characterization of the human gene encoding Ito: further diversity by alternative mRNA splicing. Am J Physiol. 1998; 275: H1963eCH1970.
An WF, Bowlby MR, Betty M, Cao J, Ling HP, Mendoza G, Hinson JW, Mattsson KI, Strassle BW, Trimmer JS, Rhodes KJ. Modulation of A-type potassium channels by a family of calcium sensors. Nature. 2000; 403: 553eC556.
Kuo HC, Cheng CF, Clark RB, Lin JJ, Lin JL, Hoshijima M, Nguyen-Tran VT, Gu Y, Ikeda Y, Chu PH, Ross J, Giles WR, Chien KR. A defect in the Kv channel-interacting protein 2 (KChIP2) gene leads to a complete loss of Ito and confers susceptibility to ventricular tachycardia. Cell. 2001; 107: 801eC813.
Rosati B, Pan Z, Lypen S, Wang HS, Cohen I, Dixon JE, McKinnon D. Regulation of KChIP2 potassium channel beta subunit gene expression underlies the gradient of transient outward current in canine and human ventricle. J Physiol. 2001; 533: 119eC125.
Deschenes I, DiSilvestre D, Juang J, Wu RC, An WF, Tomaselli GF. Regulation of Kv4.3 current by KChIP2 splice variants:a component of native cardiac Ito Circulation. 2002; 106: 423eC429.
Rosati B, Grau F, Rodriguez S, Li H, Nerbonne JM, McKinnon D. Co-ordinate patterns of KChIP2 mRNA, protein and transient outward current expression throughout canine ventricle. J Physiol. 2003; 548: 815eC822.
Dixon JE, McKinnon D. Quantitative analysis of potassium channel mRNA expression in atrial and ventricular muscle of rats. Circ Res. 1994; 75: 252eC260.
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: 1067eC1076.
Sah R, Oudit GY, Nguyen TT, Lim HW, Wickenden AD, Wilson GJ, Molkentin JD, Backx PH. Inhibition of calcineurin and sarcolemmal Ca2+ influx protects cardiac morphology and ventricular function in Kv4.2N transgenic mice. Circulation. 2002; 105: 1850eC1856.
Kassiri Z, Zobel C, Nguyen TT, Molkentin JD, Backx PH. Reduction of Ito causes hypertrophy in neonatal rat ventricular myocytes. Circ Res. 2002; 90: 578eC585.
Zobel C, Kassiri Z, Nguyen TT, Meng Y, Backx PH. Prevention of hypertrophy by overexpression of Kv4.2 in cultured neonatal cardiomyocytes. Circulation. 2002; 106: 2385eC2391.
Aimond F, Kwak S, Rhodes KJ, Nerbonne JM. Accessory Kvbeta1 subunits differentially modulate the functional expression of voltage-gated K+ channels in mouse ventricular myocytes. Circ Res. 2005; 96: 451eC458.
Sanguinetti MC, Johnson JH, Hammerland LG, Kelbaugh PR, Volkmann RA, Saccomano NA, Mueller AL. Heteropodatoxins: peptides isolated from spider venom that block Kv4.2 potassium channels. Mol Pharmacol. 1997; 51: 491eC498.
Marionneau C, Couette B, Liu J, Li H, Mangoni ME, Nargeot J, Lei M, Escande D, Demolombe S. Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart. J Physiol. 2005; 562: 223eC234.
Rosati B, McKinnon D. Regulation of ion channel expression. Circ Res. 2004; 94: 874eC883.
Deschenes I, Tomaselli GF. Modulation of Kv4.3 current by accessory subunits. FEBS Lett. 2002; 528: 183eC188.
Wong W, Newel EW, Jugloff DG, Jones OT, Schlichter LC. Cell surface targeting and clustering interactions between heterologously expressed PSD-95 and the Shal voltage-gated potassium channel, Kv4.2. J Biol Chem. 2002; 277: 20423eC20430.
Petrecca K, Miller DM, Shrier A. Localization and enhanced current density of the Kv4.2 potassium channel by interaction with the actin binding protein filamin. J Neurosci. 2000; 20: 8736eC8740.
Wang Z, Eldstrom JR, Jantzi J, Moore ED, Fedida D. Increased focal Kv4.2 channel expression at the plasma membrane is the result of actin depolymerization. Am J Physiol. 2004; 286: H749eCH759.
Ma D, Zerangue N, Lin YF, Collins A, Yu M, Jan YN, Jan LY. Role of ER export signals in controlling surface potassium channel numbers. Science. 2001; 291: 316eC319.
Ma D, Jan LY. ER transport signals and trafficking of potassium channels and receptors. Curr Opin Neurobiol. 2002; 12: 287eC292.(Weinong Guo, W. Edward Ju)
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
Program in Neurobiology (T.L.S.), Children’s Hospital, Harvard Medical School, Boston, Mass.
Abstract
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, Ito,f, channels. Biochemical studies suggest that mouse ventricular Ito,f channels reflect the heteromeric assembly of Kv4.2 and Kv4.3 with the accessory subunits, KChIP2 and Kv1, and that Kv4.2 is the primary determinant of regional differences in (mouse ventricular) Ito,f densities. Interestingly, the phenotypic consequences of manipulating Ito,f expression in different mouse models are distinct. In the experiments here, the effects of the targeted deletion of Kv4.2 (Kv4.2eC/eC) were examined. Unexpectedly, voltage-clamp recordings from Kv4.2eC/eC ventricular myocytes revealed that Ito,f is eliminated. In addition, the slow transient outward K+ current, Ito,s, and the Kv1.4 protein (which encodes Ito,s) are upregulated in Kv4.2eC/eC ventricles. Although Kv4.3 mRNA/protein expression is not measurably affected, KChIP2 expression is markedly reduced in Kv4.2eC/eC ventricles. Similar to Kv4.3, expression of Kv1, as well as Kv1.5 and Kv2.1, is similar in wild-type and Kv4.2eC/eC ventricles. In addition, and in marked contrast to previous findings in mice expressing a truncated Kv4.2 transgene, the elimination Ito,f in Kv4.2eC/eC 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 Ito,f channels but also that the loss of Ito,f per se does not have overt pathophysiological consequences.
Key Words: Ito,s Kv channels remodeling arrhythmia hypertrophy
Introduction
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 () subunits in the generation of these channels.1 In adult mouse ventricles, for example, multiple Kv currents are coexpressed.2eC10 All available evidence suggests that subunits of the Kv4 subfamily underlie fast inactivating and recovering cardiac transient outward, Ito,f, channels.1 Biochemical studies suggest that mouse ventricular Ito,f channels reflect the heteromeric assembly of Kv4.2 and Kv4.3.10 In large mammals, however, Kv4.2 is not expressed, and Ito,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 Ito,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 Ito,f is eliminated in ventricular myocytes isolated from mice in which the KChIP2 locus was disrupted.14 In canine ventricles, KChIP2 message15,16 and protein17 expression parallel variations in Ito,f densities, suggesting that KChIP2 is the primary determinant of Ito,f gradients.15,17 In rodent ventricles, however, KChIP2 is uniformly expressed, and regional differences in Ito,f densities are correlated with heterogeneities in Kv4.2 expression.10,18
Interestingly, the phenotypic consequences of manipulating Ito,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 Ito,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, Ito,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 Ito,f in neonatal rat ventricular myocytes in vitro also reportedly results in cellular hypertrophy,21 and overexpression of Kv4.2 (and upregulation of Ito,f) prevents this effect.22 Although hypertrophy was attributed to the attenuation of Ito,f in these studies,19eC22 hypertrophy is not evident in Kv4.2DN7 or in KChIP2eC/eC14 ventricles in which Ito,f is undetectable.4,7,14
The experiments here were undertaken to explore the role of Kv4.2 in the generation of mouse ventricular Ito,f channels. Voltage-clamp recordings from myocytes isolated from mice bearing a targeted disruption of the KCND2 locus (Kv4.2eC/eC) reveal that Ito,f is eliminated. Although there is no detectable effect on Kv4.3 expression, KChIP2 expression is markedly reduced in Kv4.2eC/eC ventricles. The results demonstrate that the Kv4.2 subunit is required for functional cell surface expression of mouse ventricular Ito,f channels and that the loss of Ito,f does not result in hypertrophy or compromised cardiac functioning.
Materials and Methods
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 Kv4.2eC/eC 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) Locus
In the targeting construct used to generate Kv4.2eC/eC 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 S5eCS6 (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.
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 (Kv4.2eC/eC) Mice
Blastocyst injections yielded 8 chimeric mice, 3 of which provided germline transmission on crossing with WT (FVB) mice. Congenic FVB (Kv4.2+/eC) mice were bred to establish the Kv4.2eC/eC line used here. A representative Southern blot of tail DNA from the offspring of Kv4.2+/eC 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+/eC animals, whereas no Kv4.2 message is detected in Kv4.2eC/eC brains/hearts (Figure 1D). Western blots confirmed robust expression of Kv4.2 protein in WT ventricles, whereas Kv4.2 is undetectable in Kv4.2eC/eC ventricles (Figure 1E).
The Kv4.2eC/eC mice are viable and fertile and, on gross examination, are indistinguishable from WT mice. Mean±SEM body weights in 10-week male WT and Kv4.2eC/eC 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 Kv4.2eC/eC (102±3 mg) heart weights are similar. Heart to body weight ratios are also not significantly different, and gross histological examination of Kv4.2eC/eC 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 Kv4.2eC/eC animals.
Echocardiographic analysis of (10-week) Kv4.2eC/eC 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 Kv4.2eC/eC 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 Kv4.2eC/eC Ventricular Myocytes
Peak Kv current (Ipeak) densities are significantly (P<0.001) lower in myocytes isolated from the LV apex (LVA) of Kv4.2eC/eC (Figure 2B), compared with WT (Figure 2A), animals (Table 1); IK1 densities in WT and Kv4.2eC/eC cells are indistinguishable (not illustrated). In addition, the waveforms of the currents in WT and Kv4.2eC/eC LVA myocytes are distinct (Figure 2). In particular, the rapid component of current decay, which reflects Ito,f2,3,7 and is prominent in WT LVA cells (Figure 2A), is not evident in Kv4.2eC/eC LVA cells (Figure 2B). Kinetic analysis of the decay phases of the currents indeed revealed that the rapidly inactivating current is eliminated in all Kv4.2eC/eC LVA myocytes (Table 1).
Previous studies have demonstrated that peak Kv current densities are lower and that Ito,f densities are variable in interventricular septum myocytes.2,3,7 In &75% of WT septum cells, Ito,f is expressed, whereas in the other &25%, Ito,f is undetectable.2 All septum cells, however, express a rapidly activating and slowly inactivating/recovering transient Kv current, Ito,s, that is not expressed in LVA myocytes.2,3,7 In all Kv4.2eC/eC septum cells, Ito,f is undetectable (Figure 2B). Peak Kv current densities are also reduced in right ventricular (RV) myocytes from Kv4.2eC/eC animals (supplemental Figure I), and similar to Kv4.2eC/eC LVA and septum cells, Ito,f is undetectable in Kv4.2eC/eC RV myocytes (Table 1). Indeed, the Kv currents in Kv4.2eC/eC RV (LVA and septum) cells are indistinguishable from those recorded from myocytes expressing the dominant-negative Kv4.2DN construct (supplemental Figure I), in which Ito,f is eliminated.4eC6
Slowly Inactivating Current Is Evident in Kv4.2eC/eC 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 Kv4.2eC/eC 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 Ito,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 Ito,s in WT septum cells, suggesting that elimination of Ito,f in LVA myocytes results in Ito,s upregulation. Similar results were obtained on analysis of the Kv currents in Kv4.2eC/eC RV cells (supplemental Figure I).
Another interpretation of the appearance of the slowly decaying current in Kv4.2eC/eC 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 Ito,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 Kv4.2eC/eC Ventricular Myocytes
In addition to rapid inactivation, mouse ventricular Ito,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 Ito,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 Kv4.2eC/eC myocytes (Figure 2C and 2D). As previously reported,2,3,7 recovery of Ito,f in WT LVA myocytes follows a monoexponential time course with a mean±SEM rec of 37±6 ms (Figure 2E). Recovery in Kv4.2eC/eC 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 Kv4.2eC/eC 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 Kv4.2eC/eC LVA myocytes is similar to the rec for Ito,s in WT septum cells.2,5,7 Similar results were obtained in experiments on Kv4.2eC/eC RV cells.
No Heteropodatoxin-Sensitive Currents Are Evident in Kv4.2eC/eC Myocytes
The results presented above suggest that the "novel" slowly inactivating/recovering Kv current evident in Kv4.2eC/eC 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 Ito,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 Kv4.2eC/eC 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 Ito,f. In contrast, Kv currents in Kv4.2eC/eC 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 Kv4.2eC/eC 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 Kv4.2eC/eC LVA (and RV) myocytes reflects the presence of Ito,s or some other, previously undescribed, Kv current.
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 Kv4.2eC/eC animals. As evident in the representative recordings in Figure 4A, the morphologies of the QRS complexes and P waves in WT and Kv4.2eC/eC animals are indistinguishable. In addition, there were no significant differences in the durations of the QT, PR, QRS, or RR intervals in WT and Kv4.2eC/eC mice (Table 2). Mean heart rates measured in WT and Kv4.2eC/eC animals are also indistinguishable, and mean corrected QT (QTc) intervals in 10-week Kv4.2eC/eC 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 Kv4.2eC/eC animals during 48 hours of continuous monitoring also revealed no evidence of rhythm disturbances (not illustrated).
The similarities in QT (QTc) intervals in Kv4.2eC/eC and WT animals suggest that ventricular (action potential) repolarization is not affected by the deletion of Kv4.2, in spite of the loss of Ito,f. Consistent with this hypothesis, action potential waveforms in Kv4.2eC/eC 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 Kv4.2eC/eC LVA myocytes are not significantly different (Figure 4C).
Molecular Basis of Kv Channel Remodeling in Kv4.2eC/eC Ventricles
The electrophysiological findings that Ito,f is eliminated and that there is no evidence for the presence of Kv4.3 channels in Kv4.2eC/eC ventricular myocytes were unexpected in light of previous studies demonstrating that Kv4.3 forms functional channels in heterologous systems10,12,16 and that Ito,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 Ito,s channels in WT septum cells, suggesting that Kv1.4 expression might also be affected in Kv4.2eC/eC ventricles.
Subsequent experiments were focused on exploring the hypothesis that molecular remodeling occurs in Kv4.2eC/eC 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 Ito,f, KCND3 (Kv4.3),10 KCNIP2 (KChIP2),10 and KCNAB1 (Kv1)23 are not measurably different in Kv4.2eC/eC and WT ventricles (Figure 5A). In Kv4.2eC/eC ventricles, there is a small, but statistically significant (P<0.01) increase in KCNA5 (Kv1.5), which encodes 1 component of IK,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 Kv4.2eC/eC ventricles.
To validate the TaqMan low-density array data, SYBR green quantitative RT-PCR was performed on Kv4.2eC/eC and WT ventricles using Kv subunit specific primers (supplemental Table III). These analyses also revealed no significant differences in Kv1.4, Kv4.3, Kv1, or Kv2.1 expression in Kv4.2eC/eC 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 Kv4.2eC/eC ventricles (Figure 5B). The small reduction in KCNIP2 expression is also evident in samples from 5-month-old Kv4.2eC/eC animals (Figure 5F). Taken together, these combined analyses (Figure 5 and supplemental Figure II) demonstrate very little molecular remodeling at the transcript level in Kv4.2eC/eC 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 Kv4.2eC/eC LV and RV samples. Expression of Kv1, 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 Kv4.2eC/eC (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.
Discussion
Targeted Disruption of Kv4.2 Eliminates Mouse Ventricular Ito,f
The results presented here demonstrate that Ito,f is eliminated in myocytes isolated from the left and right ventricles and from the interventricular septum of Kv4.2eC/eC mice. In addition, there are no rapidly inactivating/recovering Kv currents and no residual HpTX-3eCsensitive currents detected in Kv4.2eC/eC ventricular myocytes. There is no evidence, therefore, that Kv4.3 forms functional homomeric Kv channels in Kv4.2eC/eC mouse ventricles. The elimination of Ito,f is accompanied, however, by an increase in a slowly inactivating/recovering Kv current similar to Ito,s in WT septum cells. These observations, ie, the loss of Ito,f and the upregulation of Ito,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 Ito,f in Kv4.2eC/eC hearts is not reflected in altered action potential waveforms or electrocardiograms.
The fact that action potential waveforms and QTc intervals in Kv4.2eC/eC and WT myocytes/animals are indistinguishable suggests that the upregulation of Ito,s compensates for the loss of Ito,f. Action potential and QTc prolongation, however, are evident in Kv4.2DN mice,4,7 in which Ito,f is also eliminated and Ito,s is also upregulated. There is a statistically significant (P<0.001) difference in the magnitude of Ito,s measured in Kv4.2eC/eC RV cells compared with Kv4.2DN RV cells7 that may account, at least in part, for the phenotypic differences in the Kv4.2eC/eC and Kv4.2DN animals. In RV cells from Kv4.2eC/eC and Kv4.2DN animals, mean±SEM Ito,s densities were 15.6±1.9 pA/pF and 7.5±1.4 pA/pF, respectively. The densities of Ito,s in Kv4.2eC/eC and Kv4.2DN LVA myocytes, however, are similar, suggesting that the upregulation of Ito,s alone does not explain the normalization of action potentials in Kv4.2eC/eC 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 Ito,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 Kv4.2eC/eC Ventricles
It has been suggested that electrical remodeling (Ito,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 Kv4.2eC/eC ventricles. It is unclear whether the increase in Kv1.4 protein alone underlies the upregulation of Ito,s in Kv4.2eC/eC ventricles or, alternatively, whether there are additional factors, such as interactions with auxiliary subunits3,27 or changes in the mechanisms regulating channel trafficking and cell surface expression.1,28eC30
The finding that Kv4.3 message/protein expression is not measurably affected in Kv4.2eC/eC ventricles lacking Ito,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.28eC30 In this context, it is interesting that KChIP2 protein expression is markedly reduced in Kv4.2eC/eC ventricles. There are also modest changes in KCNIP2 expression, although it seems almost certain that the predominant effect in Kv4.2eC/eC 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 Ito,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 Ito,f channels.10 In addition, Kv4.2 is differentially expressed in mouse ventricles and differences in Kv4.2 expression parallel regional variations in Ito,f densities.10 The observation here that there are no rapidly inactivating/recovering Kv currents, as well as no HpTx-sensitive Kv currents, in Kv4.2eC/eC 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 Ito,f channels. These findings were unexpected in light of previous studies suggesting that Kv4.3 forms functional channels in heterologous systems10,12,27 and that homomeric Kv4.3 channels underlie Ito,f in large mammals, including humans.11,12 The absence of functional Ito,f channels and the loss of the KChIP2 protein in Kv4.2eC/eC ventricles suggest that additional subunits may be involved in regulating Kv4.3-encoded Ito,f channel assembly and/or trafficking in human heart.
Relationship With Previous Studies
In ventricular myocytes isolated from Kv4.2DN mice, Ito,f is selectively eliminated and action potentials and QT intervals are prolonged.4,7 In contrast, Ito,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 IK1, as well as Ito,f, densities.19 Although the pathological effects of Kv4.2N expression were attributed to attenuation of Ito,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 Kv4.2eC/eC or Kv4.2DN hearts. Clearly, the absence of pathology in Kv4.2eC/eC and Kv4.2 DN animals argues strongly against a causal relationship between the loss of Ito,f and cardiac hypertrophy, as previously suggested.19eC22
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.
These authors contributed equally to this study.
References
Nerbonne JM, Kass RS. Molecular physiology of cardiac repolarization. Physiol Rev. 2005; 85: 1205eC1253.
Xu H, Guo W, Nerbonne JM. Four kinetically distinct depolarization-activated K+ currents in adult mouse ventricular myocytes. J Gen Physiol. 1999; 113: 661eC678.
Brunet S, Aimond F, Li H, Guo W, Eldstrom J, Fedida D, Yamada KA, Nerbonne JM. Heterogeneous expression of repolarizing, voltage-gated K+ currents in adult mouse ventricles. J Physiol. 2004; 559: 103eC120.
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 alpha subunit. Circ Res. 1998; 83: 560eC567.
Guo W, Xu H, London B, Nerbonne JM. Molecular basis of transient outward K+ current diversity in mouse ventricular myocytes. J Physiol. 1999; 521: 587eC599.
Xu H, Barry DM, Li H, Brunet S, Nerbonne JM. Attenuation of the slow component of delayed rectification, action potential prolongation, and triggered activity in mice expressing a dominant-negative Kv2 alpha subunit. Circ Res. 1999; 85: 623eC633.
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 alpha subunit. Circ Res. 2000; 87: 73eC79, 2000.
London B, Guo W, Pan XH, Lee JS, Shusterman V, Logothetis DA, Nerbonne JM, Hill JA. Targeted replacement of Kv1.5 in the mouse leads to loss of the 4-aminoyridine-sensitive component of IK,slow and resistance to drug induced QT prolongation. Circ Res. 2001; 88: 940eC946.
Li H, Guo W, Yamada KA, Nerbonne JM. Selective elimination of the 4-AP-sensitive component of delayed rectification, IK,slow1, in mouse ventricular myocytes expressing a dominant negative Kv1.5 alpha subunit. Am J Physiol. 2004; 286: H319eCH328.
Guo W, Li H, Aimond F, Johns DC, Rhodes KJ, Trimmer JS, Nerbonne JM. Role of heteromultimers in the generation of myocardial transient outward K+ currents. Circ Res. 2002; 90: 586eC593.
Dixon JE, Shi W, Wang HS, McDonald C, Yu H, Wymore RS, Cohen IS, McKinnon D. Role of the Kv4.3 K+ channel in ventricular muscle. A molecular correlate for the transient outward current. Circ Res. 1996; 79: 659eC668.
Kong W, Po S, Yamagishi T, Ashen MD, Stetten G, Tomaselli GF. Isolation and characterization of the human gene encoding Ito: further diversity by alternative mRNA splicing. Am J Physiol. 1998; 275: H1963eCH1970.
An WF, Bowlby MR, Betty M, Cao J, Ling HP, Mendoza G, Hinson JW, Mattsson KI, Strassle BW, Trimmer JS, Rhodes KJ. Modulation of A-type potassium channels by a family of calcium sensors. Nature. 2000; 403: 553eC556.
Kuo HC, Cheng CF, Clark RB, Lin JJ, Lin JL, Hoshijima M, Nguyen-Tran VT, Gu Y, Ikeda Y, Chu PH, Ross J, Giles WR, Chien KR. A defect in the Kv channel-interacting protein 2 (KChIP2) gene leads to a complete loss of Ito and confers susceptibility to ventricular tachycardia. Cell. 2001; 107: 801eC813.
Rosati B, Pan Z, Lypen S, Wang HS, Cohen I, Dixon JE, McKinnon D. Regulation of KChIP2 potassium channel beta subunit gene expression underlies the gradient of transient outward current in canine and human ventricle. J Physiol. 2001; 533: 119eC125.
Deschenes I, DiSilvestre D, Juang J, Wu RC, An WF, Tomaselli GF. Regulation of Kv4.3 current by KChIP2 splice variants:a component of native cardiac Ito Circulation. 2002; 106: 423eC429.
Rosati B, Grau F, Rodriguez S, Li H, Nerbonne JM, McKinnon D. Co-ordinate patterns of KChIP2 mRNA, protein and transient outward current expression throughout canine ventricle. J Physiol. 2003; 548: 815eC822.
Dixon JE, McKinnon D. Quantitative analysis of potassium channel mRNA expression in atrial and ventricular muscle of rats. Circ Res. 1994; 75: 252eC260.
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: 1067eC1076.
Sah R, Oudit GY, Nguyen TT, Lim HW, Wickenden AD, Wilson GJ, Molkentin JD, Backx PH. Inhibition of calcineurin and sarcolemmal Ca2+ influx protects cardiac morphology and ventricular function in Kv4.2N transgenic mice. Circulation. 2002; 105: 1850eC1856.
Kassiri Z, Zobel C, Nguyen TT, Molkentin JD, Backx PH. Reduction of Ito causes hypertrophy in neonatal rat ventricular myocytes. Circ Res. 2002; 90: 578eC585.
Zobel C, Kassiri Z, Nguyen TT, Meng Y, Backx PH. Prevention of hypertrophy by overexpression of Kv4.2 in cultured neonatal cardiomyocytes. Circulation. 2002; 106: 2385eC2391.
Aimond F, Kwak S, Rhodes KJ, Nerbonne JM. Accessory Kvbeta1 subunits differentially modulate the functional expression of voltage-gated K+ channels in mouse ventricular myocytes. Circ Res. 2005; 96: 451eC458.
Sanguinetti MC, Johnson JH, Hammerland LG, Kelbaugh PR, Volkmann RA, Saccomano NA, Mueller AL. Heteropodatoxins: peptides isolated from spider venom that block Kv4.2 potassium channels. Mol Pharmacol. 1997; 51: 491eC498.
Marionneau C, Couette B, Liu J, Li H, Mangoni ME, Nargeot J, Lei M, Escande D, Demolombe S. Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart. J Physiol. 2005; 562: 223eC234.
Rosati B, McKinnon D. Regulation of ion channel expression. Circ Res. 2004; 94: 874eC883.
Deschenes I, Tomaselli GF. Modulation of Kv4.3 current by accessory subunits. FEBS Lett. 2002; 528: 183eC188.
Wong W, Newel EW, Jugloff DG, Jones OT, Schlichter LC. Cell surface targeting and clustering interactions between heterologously expressed PSD-95 and the Shal voltage-gated potassium channel, Kv4.2. J Biol Chem. 2002; 277: 20423eC20430.
Petrecca K, Miller DM, Shrier A. Localization and enhanced current density of the Kv4.2 potassium channel by interaction with the actin binding protein filamin. J Neurosci. 2000; 20: 8736eC8740.
Wang Z, Eldstrom JR, Jantzi J, Moore ED, Fedida D. Increased focal Kv4.2 channel expression at the plasma membrane is the result of actin depolymerization. Am J Physiol. 2004; 286: H749eCH759.
Ma D, Zerangue N, Lin YF, Collins A, Yu M, Jan YN, Jan LY. Role of ER export signals in controlling surface potassium channel numbers. Science. 2001; 291: 316eC319.
Ma D, Jan LY. ER transport signals and trafficking of potassium channels and receptors. Curr Opin Neurobiol. 2002; 12: 287eC292.(Weinong Guo, W. Edward Ju)