Reduction of Ito Causes Hypertrophy in Neonatal Rat Ventricular Myocytes
Prolonged action potential duration (APD) and decreased transient outward K+ current (Ito) as a result of decreased expression of Kv4.2 and Kv4.3 genes are commonly observed in heart disease. We found that treatment of cultured neonatal rat ventricular myocytes with Heteropoda Toxin3, a blocker of cardiac Ito, induced hypertrophy as measured using cell membrane capacitance and 3H-leucine uptake. To dissect the role of specific Ito-encoding genes in hypertrophy, Ito was selectively reduced by overexpressing mutant dominant-negative (DN) transgenes. Ito amplitude was reduced equally (by about 50%) by overexpression of DN Kv1.4 (Kv1.4N) or DN Kv4.2 (either Kv4.2N or Kv4.2W362F), but only DN Kv4.2 prolonged APD duration (at 1 Hz) and induced myocyte hypertrophy. This hypertrophy was prevented by coexpressing wild-type Kv4.2 channels (Kv4.2F) with the DN Kv4.2 genes, suggesting the hypertrophy is due to Ito reduction and not nonspecific effects of transgene overexpression. The hypertrophy caused by reductions of Kv4.x-based Ito was associated with increased activity of the calcium-dependent phosphatase, calcineurin, and could be prevented by coinfection with Ad-CAIN, a specific calcineurin inhibitor. The hypertrophy and calcineurin activation induced by Kv4.2N infection were prevented by blocking Ca2+ entry and excitability with verapamil or high [K+]o. Our studies suggest that reductions of Kv4.2/3-based Ito play a role in hypertrophy signaling by activation of calcineurin.
One prominent feature of diseased cardiac muscle is prolongation of action potential duration (APD). Although changes in many ionic currents have been reported in heart disease,1 reductions in transient outward K+ current (Ito) have consistently been observed in heart disease regardless of species.2 In mammalian hearts, Ito has been shown to be encoded by Kv1.4, Kv4.2, and Kv4.3 potassium channel genes, although the relative contribution of these genes varies between species.3,4⇓ While a number of changes in cardiac function and gene expression occur in diseased hearts, decreased expression of Kv4.2 and Kv4.3 genes and associated changes in both Ito density and AP profile are commonly observed in myocytes from many animal models of heart disease2,5,6⇓⇓ and human heart failure.7 Moreover, the magnitude of Ito and the level of expression of Kv4.2 and Kv4.3 channels are also reduced by both acute and long-term activation of various receptor-mediated pathways in response to neurohumoral factors known to be involved in initiating cardiac hypertrophy, such as angiotensin II and phenylephrine.8–10⇓⇓ Despite the correlation between reduced Kv4.2/3 expression and heart disease, the link between reductions in Ito, Kv4.2/3 expression, and cardiac hypertrophy is still unclear, although reductions in Ito density and Kv4.2/3 expression occur very early after myocardial infarction in rats.6 APD prolongation following Ito reduction can increase Ca2+ influx through voltage-dependent L-type Ca2+ channels (ICa,L), thereby elevating [Ca2+]i.2,11⇓ Because Ca2+ is an essential cofactor for several hypertrophy signaling pathways, including calcineurin, mitogen-activated protein kinases (MAPK), and protein kinase C,12,13⇓ it is conceivable that increased Ca2+ influx by Ito reduction might modulate hypertrophy signaling in myocardium. One particularly attractive candidate pathway, linking reductions in Ito to hypertrophy, is the Ca2+/calmodulin-activated cytoplasmic serine/threonine phosphatase calcineurin that dephosphorylates NFAT3 leading to nuclear translocation and transcriptional activation of numerous hypertrophy genes.14 Moreover, calcineurin has been shown to play an important role in triggering hypertrophy signaling.13
In this study, the connection between Ito reduction and hypertrophy was investigated in cultured neonatal rat ventricular myocytes. Using DN overexpression methods, reductions in Kv4.2/3-based Ito, but not Kv1.4-based Ito, causes myocyte hypertrophy that appears to require calcineurin activation.
Materials and Methods
Neonatal Rat Ventricular Myocyte Isolation
Neonatal Sprague-Dawley (1 to 2 days old) rat ventricular myocytes (Charles River, Montreal, Canada) were isolated and cultured as described previously.15 For adenoviral infection studies, myocytes were infected (5 to 10 PFU/cell) for 4 to 6 hours immediately following replacement of serum-containing culture medium with serum-free medium 24 hours after isolation. This titer of viral infection did not cause any detectable cell death, which is consistent with previous studies.16 Electrophysiological recordings were performed at least 30 hours after the myocytes were serum-starved and infected with adenoviruses.
Recombinant adenoviruses were generated (using pAd-Easy) by inserting the green fluorescent protein (GFP) gene with or without the K+ channel transgenes into the E1 transcription region of adenovirus backbone under a CMV promoter.17 Using this technique, we constructed adenoviruses expressing full-length Kv4.2 and Kv1.4 genes or the DN Kv4.2N (amino acids 1 to 311), Kv4.2M (Kv4.2W362F) (kindly provided by Dr J. Nerbonne, Washington University, St. Louis, Mo), and Kv1.4N (amino acids 1 to 385)18 transgenes. Viruses were purified by CsCl-gradient banding after plaque-purification. Expression of these genes or transgenes in cultured myocytes was confirmed by Western blot analysis (data not shown). The production of adenoviruses expressing CAIN (Ad-CAIN) has been described previously.19
Rhodamine-Conjugated Phalloidin Staining
Actin filaments were visualized in cultured myocytes using Rhodamine-conjugated Phalloidin and imaged using a laser scanning confocal microscope (Bio-Rad MRC 600).
Whole cell patch-clamp recordings were done as described previously15 at room temperature, 30 to 70 hours after the myocytes were serum-starved and infected with adenoviruses. Infected myocytes were identified by their GFP fluorescence.
3H-Leucine Uptake Experiments
To evaluate the rate of protein synthesis, serum-starved and infected myocytes were incubated in 1 μCi 3H-Leucine (per 2×105 cells) overnight. Next, the cells were washed with PBS, treated with Trichloroacetic acid (TCA), harvested by 0.5 mol/L NaOH, and their radioactivity was recorded using a scintillation counter.
Calcineurin phosphatase enzymatic activity was determined as described previously.20
Luciferase Assay to Measure NFAT Activity
To measure NFAT activity, as a representative of calcineurin activity, we measured luciferase activity of myocytes 48 hours after transfection with pNFAT-luc (kindly provided by Dr M. Hussain, University Health Network, Toronto, Canada). We used dual-luciferase reporter (DLR) assay, which enabled us to correct for transfection efficiency.
All averaged data are presented as mean±SEM. Statistical significance was determined using t test to compare 2 groups, and ANOVA to compare multiple groups. Statistical significance was realized at P<0.05.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
Blockade of Ito Using Pharmacological Agents
To examine the consequences of long-term Ito reduction on cellular hypertrophy, we used Heteropoda toxin-3 (HpTx3), a peptide spider toxin that blocks Ito in rat cardiac myocytes.21 Application of HpTx3 (400 nmol/L) to cultured neonatal rat ventricular myocytes (NRVM) for 30 to 70 hours increased (P<0.05) cell membrane capacitance (21.5±0.8 pF, n=10, versus 16.4±0.5 pF, n=34, in control myocytes), as well as 3H-Leucine incorporation by 23.4±8.4% (Figure 1). Long-term treatment with HpTx3 also prolonged (P<0.01) APD (APD50=206.6±27.5 versus 76.7±16.5 ms, n=6) and reduced (P<0.05) Ito amplitude (73.1±13.1 pA, n=10, versus 141.0±18.4 pA, n=34) recorded in the absence of HpTx3. Long-term treatment with HpTx3 also reduced (P<0.01) Kv4.2 protein levels (by 34.5±3.6%), although not when myocyte excitability was blocked by elevated [K+]o (data not shown). These results suggest that prolonged Ito reduction by HpTx3 induces hypertrophy as well as decreasing expression of Ito-encoding Kv4.2 protein.
Electrophysiological Effects of DN K+ Channel Transgene Overexpression
Because cardiac Ito is encoded by Kv1.4, Kv4.2, and Kv4.3 K+-channel genes,3,4,22⇓⇓ dominant-negative (DN) transgenes were overexpressed in NRVM to dissect the relative contributions of these different K+ channel families to Ito and myocyte hypertrophy. Kv1.4N overexpression was used to selectively reduce the portion of Ito encoded by members of the Kv1.x gene family, which have been directly linked to the slow component of Ito (Ito,s).23–25⇓⇓ Kv4.2/3-based Ito, which underlies the fast component of Ito (Ito,f),4,24⇓ was reduced using two DN Kv4.2 transgenes, a truncated Kv4.2 gene (Kv4.2N), and a pore mutant (Kv4.2W362F or Kv4.2M), as used previously in transgenic mice models.18,26⇓ Figure 2A shows typical Ito traces recorded from GFP-, Kv1.4N-, Kv4.2N-, or Kv4.2M-infected myocytes in response to voltage steps to +60 mV from a holding potential of −90 mV. The corresponding average Ito amplitudes (per cell) are summarized in Figure 2B. This figure shows that overexpression of Kv1.4N decreased (P<0.001) Ito amplitude by almost 50% from 180.4±10.0 pA (10.2±0.5 pA/pF, n=28) in GFP myocytes to 94.1±6.8 pA (5.5±0.4 pA/pF, n=32) by eliminating the slow-recovering component, Ito,s.27 Similar reductions (P<0.001) were observed with overexpression of Kv4.2N (85.5±10.4 pA or 3.3±0.4 pA/pF, n=15) or Kv4.2M (80.9±7.4 pA or 3.2±0.3 pA/pF, n=22), but these constructs abolished the fast-recovering component, Ito,f.27 The relative reductions of Ito by Kv1.4N versus Kv4.2N and Kv4.2M matches closely the relative proportions of Ito,s versus Ito,f that exist in NRVM.15,27⇓
Because Ito is an important current for the early repolarization of rat cardiac action potentials (APs), we anticipated that reductions of Ito would cause prolongation of APD. Typical APs recorded at 1 Hz stimulation rate from GFP-, Kv1.4N-, Kv4.2N-, and Kv4.2M-infected myocytes are presented in Figure 2C with Figure 2D summarizing averaged APD50 and APD90. Despite causing significant reductions in Ito, overexpression of Kv1.4N transgene did not alter the APD50 or APD90 (P>0.4) (97.6±13.3 and 190.4±13.6 ms, n=11) compared with GFP (84.5±13.3 and 168.2±19.9 ms, n=11). On the other hand, overexpression of Kv4.2N or Kv4.2M transgenes prolonged (P<0.001) APD50 (266.7±28.8 or 270.9±31.9 ms, n=11) as well as APD90 (362.6±30.1 or 353.7±40.8 ms, respectively). Prolongation of APD in Kv4.2N- and Kv4.2M-, but not in Kv1.4N-infected myocytes establishes that Kv1.4-based Ito,s makes minor contributions to APD15 as expected from the slow kinetics of recovery from inactivation of Ito,s (τrec,s=3 to 4 s) compared with Kv4.2/3-based Ito (τrec,f=80 to 100 ms).4,28⇓
Selective Effects of DN Channel Expression on Myocyte Hypertrophy
In addition to alterations in Ito current magnitude and APD, myocytes infected with Kv4.2N or Kv4.2M were noticeably larger than myocytes infected with either GFP or Kv1.4N when assessed using confocal microscopy (Figure 3A). Figure 3B shows that the averaged cell membrane capacitance in Kv4.2N- and Kv4.2M-infected myocytes (25.9±2.8 pF, n=19, and 24.8±0.9 pF, n=21, respectively) were larger (P<0.001) than in GFP- (16.7±0.6 pF, n=28) or Kv1.4N- (17.1±2.7 pF, n=31) infected myocytes. Moreover, Figure 3C shows that 3H-Leucine incorporation was also increased (P<0.001) following Kv4.2N (by 39.3±5.6%, n=15) or Kv4.2M (by 37.9±4.9%, n=22) infection compared with GFP- or Kv1.4N-infected myocytes. Staining NRVM with Rhodamine-conjugated phalloidin, an F-actin label, revealed that this hypertrophy did not affect the normal packing and alignment of the myofilaments (Figure 3D). On the other hand, overexpression of full-length Kv1.4 or Kv4.2 (Kv1.4F, Kv4.2F) genes did not affect cell membrane capacitance (15.3±2.5 pF, n=10, or 18.4±1.3 pF, n=11, respectively) or 3H-leucine uptake (91.9±4.1%, n=9, or 95.1±3.7%, n=12, respectively) compared with GFP-infected myocytes (P>0.3), suggesting that hypertrophy observed in Kv4.2N- and Kv4.2M-infected myocytes is related to changes in Ito levels and APD.
Because contractile activity can have major effects on cell growth and hypertrophy signaling pathways,29,30⇓ it is conceivable that Ito reductions could induce hypertrophy secondary to changing the spontaneous firing rates of AP in the NRVM. However, no difference (P>0.2) in spontaneous contraction rates were observed between the different groups (0.67±0.06 Hz in GFP-infected compared with 0.57±0.05 and 0.63±0.07 Hz in Kv4.2N- and Kv4.2M-infected myocytes). Moreover, overexpression of DN Kv4.2 in myocytes electrically paced at 2 Hz (for 48 hours) also enhanced 3H-leucine uptake (by 40.3±5.3% for Kv4.2N and by 31.5±6.2% for Kv4.2M) relative to chronically paced GFP-infected myocytes (P<0.001), despite the fact that electrical pacing alone increased (P<0.001) 3H-leucine uptake in GFP myocytes by 57.1±2.6% compared with nonpaced GFP myocytes (data not shown).
Reversal of Hypertrophy Induced By DN Kv4.2 Using Kv4.2F
To explore further whether hypertrophy induced by Kv4.2N and Kv4.2M infection was related to reductions in Ito, DN Kv4.2-infected NRVMs were coinfected with Kv4.2F. As expected, Kv4.2F coexpression increased (P<0.001) Ito amplitude (1453.4±302.4 pA, n=15 for Kv4.2N+Kv4.2F and 1129.5±271.5 pA, n=13 for Kv4.2M+Kv4.2F) and abbreviated APD (APD50=2.2±0.4 ms, APD90=10.8±3.2 ms, n=18 for Kv4.2N+Kv4.2F and APD50=2.8±0.8 and 12.4±2.1 ms, n=15 for Kv4.2M+Kv4.2F) compared with GFP, Kv4.2N or Kv4.2M infection alone. More importantly, Kv4.2F coinfection prevented increases in cell capacitance (18.9±1.0 pF, n=18 for Kv4.2N and 18.1±0.9 pF, n=15 for Kv4.2M) and 3H-leucine uptake (91.8±7.0% for Kv4.2N and 97.1±6.1% for Kv4.2M) compared with GFP-infected myocytes (P>0.4). As expected, overexpression of Kv4.2F alone elevated (P<0.001) Ito amplitude (1,808.3±473.6 pA, n=10) and shortened (P<0.001) both APD50 (2.7±0.5 ms) and APD90 (12.7±2.9 ms) at 1 Hz recording rate without affecting cell membrane capacitance or 3H-leucine uptake (as stated earlier). Taken together, these findings suggest that hypertrophy induced by Kv4.2N or Kv4.2M infection requires APD prolongation following reductions in Ito.
Hypertrophy Pathway: Calcineurin
We investigated the possible role of calcineurin in DN Kv4.2-induced hypertrophy by measuring the level of activation of NFAT (DLR assay), a calcineurin-activated transcription factor downstream from calcineurin, and by measuring the specific phosphatase activity of calcineurin (see Materials and Methods). Compared with GFP-infected myocytes, activity of NFAT was increased (P<0.001) by 68.7±8.2% in Kv4.2N- and by 66.4±10.4% in Kv4.2M-infected myocytes (n=19) (Figure 4). These increases are comparable to the 63.5±5.9% increase in calcineurin phosphatase enzymatic activity following Kv4.2N infection (data not shown). Coinfection of NRVM with Ad-CAIN (calcineurin inhibitor), a specific noncompetitive inhibitor of calcineurin,31 prevented significant increases (P>0.2) in cell capacitance (18.5±2.1 pF, n=11) (Figure 5B) and 3H-leucine uptake (97.7±3.7%) (Figure 5C), as well as changes in cell morphology (Figure 5D) in Kv4.2N-infected NRVM compared with GFP, without altering the degree of Ito reduction (81.7±17.2 pA, n=14) (Figure 5A). Overexpression of CAIN plus GFP did not measurably alter Ito density, cell capacitance, protein synthesis, or cell morphology. Similar results were found using 1000 ng/mL cyclosporine A (CsA), a nonspecific inhibitor of calcineurin32 (data not shown). These findings demonstrate that the hypertrophy response induced by Ito reduction is dependent on Ca2+-activated calcineurin.
Blockade of Myocyte Excitability and Inhibition of Ca2+ Influx
If calcineurin activation and induction of hypertrophy by DN Kv4.2 overexpression results from increased Ca2+ entry in response to APD prolongation, blockade of Ca2+ entry and excitability should prevent these effects. Application of verapamil (10 μmol/L) or high [K+]o (50 mmol/L) eliminated spontaneous beating of NRVM, and they both prevented cell hypertrophy induced by DN Kv4.2N (Figure 6). Treatment with verapamil reduced 3H-leucine uptake in Kv4.2N-infected myocytes by 41.6±6.9% (n=6) relative to untreated Kv4.2N-infected myocytes to levels indistinguishable (P>0.1) from GFP-infected myocytes in the presence of verapamil. Similarly, application of 50 mmol/L external K+ reduced 3H-leucine incorporation in Kv4.2N-infected myocytes by 36.1±2.3% (n=4) relative to untreated Kv4.2N-infected myocytes to levels that were slightly elevated (14.01±2.13%, P=0.055) over that measured in GFP-infected myocytes in the presence of 50 mmol/L external K+. Despite these effects on cell growth, verapamil and high [K+]o did not interfere with the reduction in Ito and APD prolongation after DN Kv4.2 overexpression. These observations demonstrate that cellular hypertrophy induced by DN Kv4.2 depends critically on spontaneous beating of myocytes as expected if these changes resulted from APD prolongation leading to elevated Ca2+ entry.33 Indeed, calcineurin activity and NFAT activation were both prevented in Kv4.2N-infected NRVM by verapamil and elevated [K+]o (data not shown).
Different models of cardiac disease in mammals and humans are associated with prolonged action potential duration and reduced Ito.2,7⇓ Previous studies have established that Kv1.4, Kv4.2, and Kv4.3 channels encode for Ito currents3,4,34⇓⇓ and are expressed in mammalian myocardium, although the relative contribution of these genes to Ito varies among different species in a developmental and region-specific fashion.24,35–37⇓⇓⇓ In addition, Kv1.7 channels also encode for Ito currents and are expressed in mammalian heart,23 although their contribution to cardiac Ito is unclear. In heart disease and hypertrophy, the reductions in Ito are associated with decreased Kv4.2 and Kv4.3 expression.5,6,9⇓⇓ The primary purpose of this study was to explore the possible contribution of reductions in Ito and Kv4.2/3 expression in triggering hypertrophy in cardiac myocytes.
Adenoviral infection of cultured NRVM with DN Kv4.2 and DN Kv1.4 transgenes were used to selectively eliminate currents generated by Ito-encoding cardiac genes.7 Taking advantage of the subfamily-specific nature of voltage-gated K+ channel assembly,38 Ito currents generated by Kv1.4 (and possibly Kv1.7) were reduced using a truncated Kv1.4 transgene (Kv1.4N) and those generated by KV4.2/3 channel genes were reduced using truncated (Kv4.2N) or pore-mutant (Kv4.2W362F or Kv4.2M) Kv4.2 transgenes.18,26⇓ Kv1.4N overexpression in cultured NRVM reduced Ito by about 50%, abolishing selectively slow Ito (Ito,s).27 Overexpression of Kv4.2N or Kv4.2M also reduced Ito by about 50% but selectively eliminated the fast component of Ito (Ito,f) in NRVM.27 Despite the similar reductions in Ito, only Kv4.2N/M induced measurable APD prolongation in myocytes at 1 Hz stimulation rates, a rate similar to the intrinsic beating rate of our cultured NRVM (0.55 to 0.65 Hz). These observations are anticipated because the slow rate of recovery from inactivation of Ito,s (ie, τslow=2 to 3 s) limits its contribution to membrane repolarization at 1 Hz as reported previously.15,27,39⇓⇓ By contrast, Kv4.2/3 channels recover rapidly from inactivation (τfast=80 to 100 ms)15 and are therefore expected to contribute disproportionately greater to membrane repolarization and APD in beating NRVM (discussed later).
Myocyte Hypertrophy in Response to Reductions in Kv4.2/3-Based Ito
Our studies show that chronic treatment of cultured NRVM with HpTx3, a blocker of Kv4.2 and Kv4.3 channels,21 caused myocyte hypertrophy. An interesting feature of this hypertrophy was that the endogenous Ito,f was reduced in conjunction with downregulation of Kv4.2 expression as well as APD prolongation. These results suggest that reductions in Ito and APD prolongation can induce hypertrophy in cardiac myocytes as well as reduce the expression of the Kv4.2/3-encoding Ito genes, leading to a potential positive feedback.
Consistent with our findings with HpTx3, overexpression of Kv4.2N or Kv4.2M transgenes also induced marked hypertrophy estimated by increased cell membrane capacitance, 3H-leucine uptake, or cell size in conjunction with electrical changes discussed earlier. The lack of differences between the two DN Kv4.2 strategies in NRVM was somewhat surprising because transgenic (TG) mice overexpressing the Kv4.2N transgene develop cardiac hypertrophy as well as enhanced contractility18 while mice overexpressing Kv4.2M are normal.26 The lack of differences in the response of NRVM to Kv4.2N versus Kv4.2M overexpression seems inconsistent with previous suggestions that differences in the phenotypes between the corresponding TG models results from toxic effects of the Kv4.2N transgene.40 Regardless, this hypertrophy induced by DN Kv4.2 (or HpTx3) was not due to effects of these interventions on beating rate because spontaneous beating rates were not different between Kv1.4N-, Kv4.2N-, Kv4.2M-, and GFP-infected myocytes (see Mechanism of Hypertrophy section). Furthermore, DN Kv4.2 infection also induced hypertrophy in NRVM electrically stimulated at 2 Hz. By contrast, despite reducing Ito densities by 50%, DN Kv1.4 infection did not induce cardiac hypertrophy in spontaneously beating cultured NRVMs (at about 0.6 Hz). The presence of cardiac hypertrophy with DN Kv4.2 infection, combined with the absence of hypertrophy when Kv1.4-based currents were reduced, suggest that it is APD prolongation that induces hypertrophy in beating NRVMs with reduced Ito. Indeed, elimination of Ito,s by Kv1.4N is expected to only reduce total Ito amplitude by 10% to 20% when cells are beating spontaneously versus more than 80% reduction in Ito amplitude by DN Kv4.2 infection. These observations are consistent with the results from several rodent models of cardiac disease showing that Kv4.2 expression is reduced while Kv1.4 expression is increased2,18⇓ and results in NRVM treated with phenylephrine (an α-adrenoreceptor agonist).10
Mechanism of Hypertrophy
We have demonstrated previously that reductions in Kv4.2/3-based Ito channel expression can prolong APD, increase Ca2+ influx through L-type Ca2+ channels, and elevate [Ca2+]i transients in each cardiac cycle.2,41⇓ These increases in [Ca2+]i could potentially modulate the activation of several Ca2+-activated hypertrophy pathways,13 such as PKC (α and β), MAPK pathways (SAPK, ERK, and P38), and/or calcineurin (see Molkentin and Dorn42 for review). Our studies strongly support a connection between APD prolongation, Ca2+ entry, and cell growth in our DN Kv4.2-infected myocytes for several reasons. First, blocking excitability and Ca2+ entry with verapamil or elevated [K+]o prevented the hypertrophy induced by DN Kv4.2 overexpression without averting the reductions of Ito in cultured NRVM. Moreover, 3H-leucine incorporation following blockade of excitability was normalized between GFP- and DN Kv4.2-infected myocytes and reduced to below that observed in GFP-infected NRVM without blockade. Second, a strong connection between APD prolongation and hypertrophy in NRVM following Ito reduction was further supported by the ability of Kv4.2F coinfection to elevate Ito, shorten APD, and prevent myocyte hypertrophy induced by DN Kv4.2, as well as the lack of hypertrophy in NRVM overexpressing Kv1.4N (which does not prolong APD). Third, overexpression of DN Kv4.2 transgenes in electrically stimulated cultured myocytes (at 2 Hz) increased 3H-leucine incorporation over and above that caused by electrical stimulation alone, which has been reported previously.30 The putative link between elevated Ca2+ influx and the induction of hypertrophy is consistent with the hypertrophy that occurs in TG mice overexpressing cardiac L-type Ca2+ channels43 and the Kv4.2N construct.33
Although numerous cell signaling pathways might be involved in transduction of Ito reductions into a hypertrophy signal, we initially focused on the possible role of calcineurin, a cytoplasmic phosphatase that is activated by calcium and Ca/CaM complex.44 Cardiac expression of a constitutively active form of calcineurin in TG mice caused severe cardiac hypertrophy that was blocked by pharmacological inhibition of calcineurin in vivo and in vitro.14 Calcineurin has also been shown to be involved in development of hypertrophy induced by angiotensin II, phenylephrine,19 and pressure overload.45 In addition, TG mice expressing inhibitory domain of either CAIN or AKA79 (calcineurin inhibiting peptides) partially reduced catecholamine- and pressure overload–induced cardiac hyertrophy.46 These and several other studies establish that calcineurin is a strong candidate contributing to cardiac myocyte hypertrophy47 possibly by integrating with other signaling pathways.48 Consistent with a prominent role for calcineurin in hypertrophy, NFAT activity and calcineurin’s phosphatase activity was increased after overexpression of Kv4.2N or Kv4.2M. Treatment with the nonspecific calcineurin inhibitor, cyclosporin A (CsA) (data not shown), or coinfection with the specific peptide inhibitor of calcineurin, CAIN,31 completely blocked the increases in NFAT activity as well as the hypertrophy in NRVM infected with DN Kv4.2 genes, suggesting that these increases in NFAT3 activity were the consequence of increased calcineurin activity with no, or minimal, interference from other hypertrophy pathways. These results are also consistent with our findings in TG mice expressing Kv4.2N,18 where cardiac hypertrophy and fibrosis were prevented and cardiac function maintained when treated with CsA or verapamil.33
It is conceivable that infection of myocytes with recombinant adenoviruses might be linked to nonspecific or toxic actions in NRVM. Indeed, increased cell death can occur when cultured myocytes are infected with high titers of adenoviruses.16 For this reason, myocytes in our studies were infected with 5 to 10 PFU/cell of purified virus for 4 to 6 hours and experiments were done 30 to 70 hours after infection. This titer consistently resulted in greater than 90% transfection efficiency with no detectable cell death as reported previously.16 In studies involving coinfection with Kv4.2N or Kv4.2M along with full length Kv4.2F, the number of PFUs per cell was increased (15 to 20 PFU/cell) and produced no evidence of cell death. More importantly, coinfection with Kv4.2F elevated Ito, shortened APD, and prevented the myocyte hypertrophy induced by DN Kv4.2. Although Ito was increased well above the control levels following Kv4.2F coinfection, attempt to reduce the Ito levels by reducing viral titers below 5 to 10 PFU/cell (of each construct) resulted in reduction in infection efficiency, making interpretation of our studies on cell populations problematic. Regardless, the ability of Kv4.2F to prevent hypertrophy suggests that the hypertrophy was not due to nonspecific or toxic effects of these viruses, or that hypertrophy induced by nonspecific effects of overexpression depends critically on APD prolongation.
In conclusion, our results show that reductions in Ito produced by Kv4.2/3 channel genes can trigger hypertrophy in NRVM by prolonging APD, which appears to depend critically on calcineurin activation. The extent to which these observations are relevant to other species, with AP profiles different from NRVMs, is uncertain and clearly warrants further investigation.
This study was supported by an operating grant (P.H.B.) from the Canadian Institute of Health Research (CIHR). Z.K. is funded by the Heart and Stroke Foundation of Canada/CIHR partnership Fund. C.Z. is funded by VERUM Foundation for Behavior and Environment and Deutsche Forschungsgemeinschaft (Zo 112/1-1). P.H.B. is a Career Investigator of Heart and Stroke Foundation of Ontario. We are grateful for equipment support from the Tiffin Trust Fund, the University of Toronto, and the Heart and Stroke/Richard Lewar Center of Excellence. The authors would like to thank Dr Jean Nerbonne for the kind gift of Kv4.2W362F cDNA. We also acknowledge NPS Pharmaceuticals for the kind gift of HpTx3.
Original received August 24, 2001; revision received January 18, 2002; accepted January 22, 2002.
- ↵Cerbai E, Barbieri M, Li Q, Mugelli A. Ionic basis of action potential prolongation of hypertrophied cardiac myocytes isolated from hypertensive rats of different ages. Cardiovasc Res. 1994; 28: 1180–1187.
- ↵Takimoto K, Li D, Hershman KM, Li P, Jackson EK, Levitan ES. Decreased expression of Kv4.2 and novel Kv4.3 K+ channel subunit mRNAs in ventricles of renovascular hypertensive rats. Circ Res. 1997; 81: 533–539.
- ↵Kaab S, Dixon J, Duc J, Ashen D, Nabauer M, Beuckelmann DJ, Steinbeck G, McKinnon D, Tomaselli GF. Molecular basis of transient outward potassium current downregulation in human heart failure: a decrease in Kv4.3 mRNA correlates with a reduction in current density. Circulation. 1998; 98: 1383–1393.
- ↵Zhang TT, Takimoto K, Stewart AF, Zhu C, Levitan ES. Independent regulation of cardiac Kv4.3 potassium channel expression by angiotensin II and phenylephrine. Circ Res. 2001; 88: 476–482.
- ↵Wickenden AD, Kaprielian R, Kassiri Z, Tsoporis JN, Tsushima R, Fishman GI, Backx PH. The role of action potential prolongation and altered intracellular calcium handling in the pathogenesis of heart failure. Cardiovasc Res. 1998; 37: 312–323.
- ↵Olson EN, Molkentin JD. Prevention of cardiac hypertrophy by calcineurin inhibition: hope or hype? Circ Res. 1999; 84: 623–632.
- ↵O’Donnell JM, Sumbilla CM, Ma H, Farrance IK, Cavagna M, Klein MG, Inesi G. Tight control of exogenous SERCA expression is required to obtain acceleration of calcium transients with minimal cytotoxic effects in cardiac myocytes. Circ Res. 2001; 88: 415–421.
- ↵He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A. 1998; 95: 2509–2514.
- ↵Wickenden AD, Lee P, Sah R, Huang Q, Fishman GI, Backx PH. Targeted expression of a dominant-negative Kv4.2 K+ channel subunit in the mouse heart. Circ Res. 1999; 85: 1067–1076.
- ↵Taigen T, De Windt LJ, Lim HW, Molkentin JD. Targeted inhibition of calcineurin prevents agonist-induced cardiomyocyte hypertrophy. Proc Natl Acad Sci U S A. 2000; 97: 1196–1201.
- ↵Lim HW, De Windt LJ, Steinberg L, Taigen T, Witt SA, Kimball TR, Molkentin JD. Calcineurin expression, activation, and function in cardiac pressure-overload hypertrophy. Circulation. 2000; 101: 2431–2437.
- ↵Sanguinetti MC, Johnson JH, Hammerland LG, Volkmann RA, Saccomano NA, Mueller AL. Heteropodatoxins: peptides isolated from spider venom that block Kv4.2 potassium channels. Mol Pharmacol. 1997; 51: 491–498.
- ↵Kalman K, Nguyen A, Tseng-Crank J, Gutman GA, Chandy KG. Genomic organization, chromosomal localization, tissue distribution, and biophysical characterization of a novel mammalian Shaker-related voltage-gated potassium channel, Kv1.7. J Biol Chem. 1998; 273: 5851–5857.
- ↵Barry DM, Xu H, Schuessler RB, Nerbonne JM. Functional knockout of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4 α-subunit. Circ Res. 1998; 83: 560–567.
- ↵Kassiri Z, Hajjar R, Backx PH. Molecular components of transient outward K+ current in cultured neonatal rat ventricular myocytes. J Mol Med. In press.
- ↵Xu H, Guo W, Nerbonne JM. Four kinetically distinct depolarization-activated K+ currents in adult mouse ventricular myocytes. J Gen Physiol. 1999; 113: 661–678.
- ↵Xia Y, McMillin JB, Lewis A, Moore M, Zhu WG, Williams RS, Kellems RE. Electrical stimulation of neonatal cardiac myocytes activates the NFAT3 and GATA4 pathways and up-regulates the adenylosuccinate synthetase 1 gene. J Biol Chem. 2000; 275: 1855–1863.
- ↵Lai MM, Burnett PE, Wolosker H, Blackshaw S, Snyder SH. CAIN, a novel physiologic protein inhibitor of calcineurin. J Biol Chem. 1998; 273: 18325–18331.
- ↵Sah R, Oudit GY, 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. 2001; 104 (suppl II): II-36.Abstract..
- ↵Yeola SW, Snyders DJ. Electrophysiological and pharmacological correspondence between Kv4.2 current and rat cardiac transient outward current. Cardiovasc Res. 1997; 33: 540–547.
- ↵Wang Z, Feng J, Shi H, Pond A, Nerbonne JM, Nattel S. Potential molecular basis of different physiological properties of the transient outward K+ current in rabbit and human atrial myocytes. Circ Res. 1999; 84: 551–561.
- ↵Xu J, Yu W, Jan YN, Jan LY, Li M. Assembly of voltage-gated potassium channels: conserved hydrophilic motifs determine subfamily-specific interactions between the α-subunits. J Biol Chem. 1995; 270: 24761–24768.
- ↵Guo W, Li H, London B, Nerbonne JM. Functional consequences of elimination of Ito,f and Ito,s: early afterdepolarizations, atrioventricular block, and ventricular arrhythmias in mice lacking Kv1.4 and expressing a dominant-negative Kv4 α-subunit. Circ Res. 2000; 87: 73–79.
- ↵Brooksby P, Levi AJ, Jones JV. Investigation of the mechanisms underlying the increased contraction of hypertrophied ventricular myocytes isolated from the spontaneously hypertensive rat. Cardiovasc Res. 1993; 27: 1268–1277.
- ↵Muth JN, Yamaguchi H, Mikala G, Grupp IL, Lewis W, Cheng H, Song LS, Lakatta EG, Varadi G, Schwartz A. Cardiac-specific overexpression of α1 subunit of L-type voltage-dependent Ca2+ channel in transgenic mice: loss of isoproterenol-induced contraction. J Biol Chem. 1999; 274: 21503–21506.
- ↵Meguro T, Hong C, Asai K, Takagi G, McKinsey TA, Olson EN, Vatner SF. Cyclosporine attenuates pressure-overload hypertrophy in mice while enhancing susceptibility to decompensation and heart failure. Circ Res. 1999; 84: 735–740.
- ↵De Windt LJ, Lim HW, Bueno OF, Liang Q, Delling U, Braz JC, Glascock BJ, Kimball TF, del Monte F, Hajjar RJ, Molkentin JD. Targeted inhibition of calcineurin attenuates cardiac hypertrophy in vivo. Proc Natl Acad Sci U S A. 2001; 98: 3322–3327.
- ↵Haq S, Choukroun G, Lim H, Tymitz KM, del Monte F, Gwathmey J, Grazette L, Michael A, Hajjar R, Force T, Molkentin JD. Differential activation of signal transduction pathways in human hearts with hypertrophy versus advanced heart failure. Circulation. 2001; 103: 670–677.