Prevention of Hypertrophy by Overexpression of Kv4.2 in Cultured Neonatal Cardiomyocytes
Background— Prolonged action potentials (APs) and decreased transient outward K+ currents (Ito) are consistent findings in hypertrophic myocardium. However, the connection of these changes with cardiac hypertrophy is unknown. The present study investigated the effects of changes in Ito and the associated alterations in AP on myocyte hypertrophy induced by phenylephrine.
Methods and Results— Chronic incubation of cultured neonatal ventricular rat myocytes (NVRMs) with phenylephrine (PE) reduced Ito density and prolonged AP duration, leading to a 2-fold increase in the net Ca2+ influx per beat and a 1.4-fold increase in Ca2+-transient amplitude. PE treatment of chronically paced (2-Hz) NVRM also induced increases in cell size, protein/DNA ratio, atrial natriuretic factor mRNA expression, as well as β/α myosin mRNA ratio. These hypertrophic changes were associated with a 2.4-fold increase in activation of nuclear factor of activated T-cells (NFAT), indicating increased activity of the Ca2+-dependent phosphatase calcineurin. Overexpression of Kv4.2 channels using adenovirus prevented the AP duration prolongation as well as the increases in Ca2+ influx and Ca2+-transient amplitude induced by PE. Kv4.2 overexpression also prohibited the PE-induced increases in cell size, protein/DNA ratio, atrial natriuretic factor expression, β/α myosin mRNA ratio, and NFAT activation.
Conclusions— Our results demonstrate that PE-mediated hypertrophy in NRVMs seems to require Ito reductions and AP prolongation associated with increased Ca2+ influx and Ca2+ transients as well as calcineurin activation. The clinical implications of these studies and the possible involvement of other signaling pathways are discussed.
Received March 28, 2002; revision received July 31, 2002; accepted August 2, 2002.
Cardiac hypertrophy is a crucial compensatory mechanism for normalizing wall stress in many forms of heart disease. Nevertheless, chronic activation of hypertrophic pathways is believed to contribute to heart disease progression toward heart failure. A consistent finding in cardiac hypertrophy is electrical remodelling and, in particular, action potential (AP) prolongation.1–3⇓⇓ Reductions in Ito density have been linked to altered AP profiles as well as slowed early repolarization4 and occur early in cardiac disease, suggesting a possible role for Ito changes in hypertrophic process.5 On a molecular level, cardiac Ito is generated by Kv4.2, Kv4.3, and Kv1.4 genes, with their relative contribution varying between species and different regions of the heart.6 However, only reductions in Kv4.2 and Kv4.3 expression have been linked consistently to the diminished Ito densities observed in cardiac hypertrophy.7
AP prolongation after reduced Ito densities can lead to increases in Ca2+ influx through L-type Ca2+ channels, resulting in elevated intracellular Ca2+ levels.7,8⇓ Because Ca2+ is an essential cofactor for hypertrophic signaling,9 it is conceivable that reductions in Ito density play a significant role in mediating cardiac hypertrophy. Indeed, we have recently demonstrated that reductions of Ito encoded by Kv4.2/3 cause hypertrophy in cultured neonatal ventricular rat myocytes (NVRMs).10 The α1-adrenoceptor agonist phenylephrine (PE) has been commonly used to induce hypertrophy in NVRM.11 Furthermore, exposure of NVRM to PE results in a reduction of Ito density and AP prolongation12 as well as reduced expression levels of Kv4.213 and Kv4.3.14 The implications of these changes in Ito on the induction of hypertrophy through PE are not known. Therefore, the present study was designed to investigate whether prevention of the reductions in Ito could inhibit the hypertrophic effects of chronic PE treatment.
Construction of Recombinant Adenoviruses
Recombinant adenoviruses were generated15 to overexpress either the green fluorescence protein (GFP) alone (AdGFP) or in combination with the Kv4.2 K+ channels (AdGFPKv4.2). Both viruses were plaque purified, and virus titers were determined using the 50% tissue culture infectious dose (TCID50) method: AdGFP 6.3×1011/mL and AdGFPKv4.2 3.95×1010/mL
Neonatal Rat Ventricular Myocyte Isolation
NVRMs were isolated and cultured as described previously.10 For patch-clamp recording experiments, 1.5×105 myocytes were plated on laminin-coated coverslips in 35-mm culture dishes. To assess the hypertrophic effects of PE treatment, 9.6×106 myocytes were split equally into 4-well culture dishes (Nunc). After 24 hours in culture, the medium was replaced by serum-free medium and virus infections were performed (AdGFP 5 TCID50/myocyte or 2 TCID50/myocyte and AdGFPKv4.2 5 TCID50/myocyte). Typically, 80% to 90% of the myocytes showed expression of GFP 24 hours after infection. PE in a concentration of 10 μmol/L was added when required. To stimulate and synchronize myocyte contraction, alternating rectangular-wave electrical pulses (50 V) of 5-ms duration at 2 Hz were applied via platinum wires that were submersed at opposite ends of each well in the 4-well culture dishes, as described previously.16
Electrophysiological recordings were performed 48 hours after serum withdrawal using borosilicate glass micropipettes (3 to 4 MOhm). Myocytes were perfused with drug-free bath solution for at least 15 minutes before measurements were performed. Whole-cell currents were filtered at 2 kHz (Axon 200A amplifier; Axon Instruments, Inc). Ito was measured at 21°C in an extracellular solution containing (in mmol/L) NaCl 140, KCl 4, CaCl2 2, MgCl2 1, CdCl2 0.5, HEPES 10, and glucose 10, pH 7.4. The intracellular solution contained (in mmol/L) KCl 140, MgCl2 1, EGTA 10, HEPES 10, and MgATP 5, pH 7.25. APs were acquired at 32°C in the same extracellular solution as above with the exemption of CdCl2. The pipette solution for the AP studies contained (in mmol/L) KCl 140, MgCl2 1, HEPES 5, and Na2ATP 5, pH 7.25. Ca2+ currents were recorded with a bath solution containing (in mmol/L) TMA-OH− 130, aspartic acid 130, HEPES 10, glucose 10, CaOH2− 1.8, and MgOH2− 1, pH 7.4. The pipette solution for ICa measurements contained (in mmol/L) CsOH 150, aspartic acid 120, EGTA 10, HEPES 10, TEA-Cl 10, and Mg2+ATP 5, pH 7.3. In voltage-clamp studies, cells were stimulated with either step depolarizations or AP clamps using typical APs recorded under current clamp conditions. Junction potentials were −4.9 mV for the Ito measurements (uncorrected) and −11.1 mV for the ICa measurements (corrected).
Ca2+ transients were measured essentially as described earlier17 using Indo-1 at 30°C in a heating chamber. Autofluorescence was measured before myocytes were loaded with Indo-1AM dye, and 405/485-nm ratios were calculated after autofluorescence subtraction following stimulation of the cardiomyocytes at 1 Hz with a Grass S44 stimulator.
Confocal images of NRVM cultured in 4-well dishes were obtained using a Bio-Rad MRC 600 laser-scanning confocal microscope with a 50× water immersion objective lens.
Measurement of Protein-DNA Ratio
Protein-DNA ratios were measured as described previously.16
RNAase Protection Assay
After 48 hours of chronic stimulation, total RNA was isolated using the RNAeasy Mini Kit (Qiagen). Antisense riboprobes for α/β myosin mRNA and atrial natriuretic factor (ANF) (both probes were a gift from Dr T.G. Parker, University Health Network, Toronto, Canada) were labeled with biotin-16-uridine-5′-triphosphate (Boehringer Mannheim) by in vitro transcription with T7 (α/β myosin) or SP6 (ANF, GAPDH) RNA polymerase of appropriate RNA synthesis vectors. The probes were hybridized with total RNA isolated as described above, and RNAase-resistant hybrids were recovered using the RPA III Kit (Ambion), analyzed on 8 mol/L urea 6% polyacrylamide sequencing gels, and visualized with the BrightStar Biodetect Kit (Ambion).
Luciferase Assay to Measure Calcineurin Activity
To assess calcineurin activity, a luciferase assay was applied as described previously.10
All data are expressed as mean±SEM. Statistical significance was determined using the unpaired or paired t test while comparing 2 groups and ANOVA to compare multiple groups.
Relationship Between AP Duration, Ito, ICa, and Ca2+ Transients
Figure 1 shows typical Ito traces, APs, and Ca2+ transients from cultured neonatal myocytes before and 5 minutes after application of 10 μmol/L PE. Both Ito density and AP duration50 (APD50) were slightly (but significantly) altered, whereas APD90 was unchanged by PE application. By contrast, chronic exposure to PE reduced Ito density by ≈70% while increasing APD50 by ≈10-fold and APD90 by ≈ 4-fold (Figure 2). Despite the large differences in the effects of PE on Ito and AP between acute and chronic applications, the effects of PE on Ca2+-transient amplitudes were quite similar (Figures 1 and 2⇓), suggesting that the acute effects of PE on APD and Ca2+ handling are distinct from its chronic effects, as suggested previously.12
As expected from previous studies,7,18⇓ the integrated Ca2+ influx per beat increased ≈2-fold in PE-treated myocytes stimulated with their own prolonged AP compared with nontreated myocytes stimulated with their corresponding typical short, control AP (Figure 3), despite reductions (P<0.01) in the ICa peak (Table). These differences in net Ca2+ influx seem to result from variations in AP duration, because the net Ca2+ influx in PE-treated myocytes became indistinguishable from untreated myocytes after depolarization with typical control APs (Figures 3B and 3E, Table). Moreover, application of typical long APs obtained from PE-treated myocytes to AdGFP-infected control cells produced ICa profiles and integrated Ca2+ influxes matching closely those recorded from PE-treated cells stimulated with similar long APs (Figures 3A and 3D, Table). As expected,7,18,19⇓⇓ increased Ca2+ entry via ICa in PE-treated myocytes was associated with elevated (P<0.05) Ca2+ transient amplitudes compared with untreated NVRM (Figures 1 and 2⇑).
Overexpression of Kv4.2 abbreviated AP durations and prevented the increases in Ca2+ influx and Ca2+ transient amplitude resulting from PE treatment (Figures 2 and 3⇑C, Table). These effects of Kv4.2 overexpression did not seem to be related to alterations in the density and biophysical properties of ICa itself, because ICa in PE-treated NRVMs was not different between AdKv4.2-infected versus AdKv4.2-uninfected myocytes when stimulated with similar prolonged AP (Figures 3D and 3F and Table). Thus, although PE might affect the expression and activity of many Ca2+-handling proteins in NVRM, the alterations in Ito and AP profile seem to be necessary for the increases in ICa and Ca2+ transient amplitude induced by chronic PE treatment.
Effects of APD on Myocyte Hypertrophy
Next, the effects of Kv4.2 overexpression on hypertrophy induced by PE were investigated. Because beating rates can influence hypertrophic signaling as well as gene expression in cultured NRVM,16 and because PE treatment20 or overexpression of Kv4.2 can conceivably affect spontaneous beating rates, NVRMs were chronically paced for 48 hours at 2 Hz. The addition of PE for 48 hours to AdGFP-infected myocytes increased cell size (Figure 4). These alterations in cell size were accompanied by increases (P<0.05) in the protein/DNA ratio, enhanced expression of the ANF, and increases in the β/α myosin heavy chain (MHC) ratio compared with nontreated AdGFP-infected myocytes (Figures 5A and 6⇓), demonstrating that PE is capable of enhancing hypertrophy in chronically paced cells. More important, Kv4.2 overexpression prevented both the increase in cell size induced by PE (Figure 4) and the increases in the protein/DNA ratio (Figure 5A), which were not related to differences in the amount of DNA levels (Figure 5B). Overexpression of Kv4.2 in PE-treated myocytes also prevented the increase in the β/α MHC ratio (P>0.05) and attenuated the increase in ANF mRNA levels (Figure 6). Collectively, these results establish that there is a strong link between AP prolongation and hypertrophy induced by PE.
Consistent with previous studies showing that the calcium-dependent phosphatase calcineurin is involved in cardiac hypertrophy21 and in PE-induced hypertrophy of cultured NRVMs,22 the level of activation of NFAT, a calcineurin-activated transcription factor, was increased (P<0.05) by PE, whereas overexpression of Kv4.2 prevented this increase of NFAT activity (activity normalized to AdGFP-infected myocytes: AdGFP+PE 242.3±34.5% versus AdKv4.2+PE 110.5±28.47%).
The present study investigated the contribution of alterations in Ito density and corresponding changes in AP profile on the development of myocyte hypertrophy induced by PE. To separate the contribution of changes in APD on the hypertrophic effects of PE treatment and the blockade of this hypertrophy by Kv4.2 overexpression from possible secondary effects of these interventions on intrinsic beating rates of NRVM (which can indirectly affect hypertrophy16), our studies were performed using cultured NRVMs electrically paced at 2 Hz. As observed previously in nonpaced NRVMs, PE treatment of paced NRVM increased cell sizes, protein/DNA ratios, and ANF mRNA levels as well as the β/α MHC mRNA ratio. Unlike in nonpaced and spontaneously beating NRVM,11,23⇓ the relative increase in ANF mRNA expression induced by PE was remarkably modest in paced myocytes.17,24⇓ This observation is probably related to the previous finding that rapid electrical pacing itself elevates ANF mRNA expression to levels comparable with those induced by PE treatment of spontaneously beating NRVMs, albeit through the activation of different signaling pathways.24
Acute exposure of NVRMs to PE resulted in relatively minor reductions in Ito and APD prolongation, as reported previously.25 By contrast, treatment of paced NRVM for 48 hours with PE caused marked reductions of Ito and associated APD prolongation that seemed to originate from reduced functional expression of Kv4.2/3, as reported previously.12–14⇓⇓ These effects in chronically treated myocytes were largely unrelated to acute effects of PE, because our electrical recordings were made in the absence of PE.
The ability of Kv4.2 overexpression to prevent hypertrophy in our studies establishes that Ito reductions and APD prolongation are prerequisites for PE-mediated hypertrophy in NRVM. However, these observations do not establish that Ito reductions by PE are the primary cause of the observed hypertrophy. The issue of whether reductions in Ito can induce hypertrophy is controversial and seems to be model-dependent. In transgenic mice, elimination of Kv4.x-based Ito and AP prolongation may29,30⇓ or may not39–41⇓⇓ lead to notable structural alterations of the heart, possibly depending on the specific dominant-negative strategy used. On the other hand, Kv4.x-based Ito reduction does cause hypertrophy in NRVMs10 requiring AP prolongation. The reasons for these inconsistencies are unclear. It is conceivable that adenoviral infections or excessive overexpression of exogenous dominant-negative proteins used to reduce Ito10,29,30⇓⇓ could stimulate hypertrophic signaling pathways that could be amplified or modulated by the associated reductions in Ito and APD prolongation, as occurs in NRVMs treated with PE. Modulation of hypertrophy by Ito reductions would explain the observations that the amount of hypertrophy induced by PE is considerably greater in nonpaced, than in paced, NRVMs10 (C. Zobel, unpublished data, 2001) and that hypertrophy is enhanced in Kv4.2W362FxKv1.4−/− mice subjected to pressure overload.42 Alternatively, Ito reductions might be sufficient for inducing hypertrophy in NRVMs and the developing heart owing to an increased propensity for growth in young myocytes. Regardless, more studies are clearly warranted to test further the modulatory effects of Ito on myocyte hypertrophy.
APD prolongation induced by chronic PE treatment was accompanied by a 2-fold increase in total Ca2+ influx per beat and a 1.4-fold increase in Ca2+ transient amplitude compared with untreated NVRM. The increases in Ca2+ influx seen with PE are clearly and directly linked to AP prolongation and not to changes in the number of active Ca2+ channels, because the application of a control AP to PE-treated NVRM resulted in Ca2+ currents indistinguishable from control cells and vice versa. The absence of measurable changes in ICa was somewhat unexpected, because chronic α1-adrenoceptor stimulation with PE has previously been shown to reduce Ca2+ current densities by 50% in cultured NVRM.12 On the other hand, another study showed that chronic α1-adrenoceptor stimulation increased L-type Ca2+ current densities.25 The reasons for these discrepancies are unclear and will require additional studies.
Chronic PE treatment was associated with hypertrophy and a 2.4-fold activation of calcineurin (whose Ca2+/calmodulin-dependent phosphatase activity was measured using NFAT activity), both of which were prevented by AP abbreviation. These results confirm an important role for calcineurin in PE-mediated hypertrophy of NVRM, as reported previously,22 but additionally suggest that AP prolongation and elevated Ca2+ are required for calcineurin activation after PE treatment. The mechanism for activation of calcineurin in our study could be linked to local increases of [Ca2+]i, particularly in the vicinity of Ca2+ channels, as a result of observed increases in Ca2+ entry after AP prolongation, because calcineurin has been shown to colocalize with L-type Ca2+ channels via the A-kinase anchoring protein AKAP79.26 Moreover, increased ICa has been shown to induce hypertrophy in NRVM treated with Ca2+ channel agonists27 as well as in transgenic mice with cardiac overexpression of either L-type Ca2+ channels28 or dominant-negative Kv4.2 channels that eliminate fast Ito.29 This mechanism is also consistent with the observation that treatment with blockers of ICa can prevent the hypertrophy induced by AP prolongation.10,20,30⇓⇓ Alternatively, the activation of calcineurin might also originate from global elevations in peak systolic [Ca2+]i; although this cannot be ruled out, phospholamban knockout mice show marked increases in Ca2+ transient amplitudes without developing cardiac hypertrophy.31
Of course, other hypertrophic signaling pathways and mechanisms, in addition to calcineurin, might also contribute to the PE-induced hypertrophy. For example, PE has been shown to activate protein kinase (PKC) ε and δ in cultured NVRM33 as well as members of the mitogen-activated protein kinase (MAPK) family,34–36⇓⇓ which are Ca2+-dependent pathways that might also be modulated by changes in AP profile and Ca2+ cycling. However, neither overexpression of constitutively active PKCε nor PKCδ induces hypertrophy in NVRM,37 whereas hypertrophy induced in cultured NRVM by PE22 and other stimuli38 require the activation of the calcineurin pathway. Alternatively, it is also possible that elevated Ca2+ transients after PE treatment will increase mechanical activity, which could stimulate cell growth32 independent of Ca2+-dependent hypertrophic signaling pathways.
Regardless of the connection between elevated Ca2+ as a result of APD prolongation with hypertrophy and calcineurin activation after PE treatment, it is clear that overexpression of Kv4.2 channels and associated increased Ito current can prevent these PE-induced changes. This suggests that the reductions in Ito currents generated by Kv4.x channels12–14⇓⇓ are necessary ingredients in the hypertrophic response induced by PE. Consequently, it is certainly possible that overexpression of other K+ channel genes besides Kv4.2-based Ito could also inhibit PE-induced hypertrophy in NRVMs. However, the predominant effect of chronic PE treatment on membrane currents is the reduction of Ito with little change in other K+ currents. In addition, because of its rapid gating kinetics compared with many other K+ channels, Ito more strongly influences early repolarization and, thereby, the amount of Ca2+ entry through ICa per beat and Ca2+ transient amplitudes.7,8,18,46⇓⇓⇓
It is possible that Kv4.2 overexpression inhibits growth in NRVMs due to nonspecific effects of the expression of this K+ channel, independent of effects on AP profile. Although this possibility is difficult to rule out, it seems unlikely, because overexpression of the full-length dominant-negative Kv4.2 mutant (Kv4.2W363F), which apparently is processed in a manner similar to the wild-type Kv4.2 protein,39 induces hypertrophy in NRVMs.10 In non–PE-treated myocytes, cell growth was also significantly inhibited (≈15%) by overexpression of Kv4.2 and APD abbreviation compared with GFP-infected myocytes (data not shown), which might, at first glance, suggest a nonspecific effect of AdKv4.2 infection. However, this mild inhibitory effect on hypertrophy is consistent with our previous studies showing that Ito reductions alone can induce hypertrophy in NRVMs,10 which is anticipated from the link between APD duration, Ca2+ cycling, and calcineurin activation. This interpretation is also consistent with previous studies (and our unpublished data) establishing that pacing itself affects myocyte growth in the cultured NRVMs.17,24⇓
The relevance of our NRVM results to hypertrophy in intact adult myocardium and human disease remains unclear for several reasons. In rodents, Ito represents the major repolarizing current, while making a relatively smaller contribution to repolarization in larger species and humans,43,44⇓ leading to very different consequences of changes in Ito amplitude.45 For example, reduced Ito in rodents leads to enhanced excitation-contraction coupling and contraction,7,8⇓ whereas the opposite occurs in larger mammals,43–45⇓⇓ which can be traced to variability in the effects of Ito on ICa.46 In addition, as already mentioned, the responsiveness of cultured NRVMs to various hypertrophic stimuli and reduction in Ito may differ markedly from that observed in intact myocardium or adult myocytes.
In summary, our results suggest that AP prolongation as a result of Ito reduction plays a crucial role in cardiac hypertrophy of NVRM after chronic α1-adrenergic receptor activation, possibly by modulating calcineurin activity in response to altered Ca2+ cycling.
This study was supported by the Canadian Institutes for Health Research (Dr Backx). Dr Zobel is supported by VERUM Foundation for Behavior and Environment and Deutsche Forschungsgemeinschaft (Zo 112/1-1). Dr Kassiri is funded by HSFC/CHIR partnership fund. Dr Backx is a Career Investigator of the Heart and Stroke Foundation of Ontario. We are grateful for equipment support from the Heart and Stroke/Richard Lewar Center for Cardiovascular Research.
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