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(Circulation Research. 2002;90:578.)
© 2002 American Heart Association, Inc.

Cellular Biology

Reduction of I_to Causes Hypertrophy in Neonatal Rat Ventricular Myocytes

Zamaneh Kassiri, Carsten Zobel, The-Tin T. Nguyen, Jeffery D. Molkentin, Peter H. Backx

From the Departments of Physiology and Medicine (Z.K., C.Z., T.-T.T.N., P.H.B.), Heart and Stroke/Richard Lewar Center and Division of Cardiology, University Health Network, University of Toronto, Toronto, Canada; and the Division of Molecular Cardiovascular Biology (J.D.M.), Children’s Hospital Medical Center, Cincinnati, Ohio.

Correspondence to Peter H. Backx, DVM, PhD, or Carsten Zobel, MD, Heart and Stroke/Richard Lewar Center, Room 68, Fitzgerald Building, University of Toronto, 150 College St, Toronto, Ontario, Canada, M5S 3E2. E-mail p.backx{at}utoronto.ca or carsten.zobel@gmx.de

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Prolonged action potential duration (APD) and decreased transient outward K⁺ current (I_to) as a result of decreased expression of K_v4.2 and K_v4.3 genes are commonly observed in heart disease. We found that treatment of cultured neonatal rat ventricular myocytes with Heteropoda Toxin₃, a blocker of cardiac I_to, induced hypertrophy as measured using cell membrane capacitance and ³H-leucine uptake. To dissect the role of specific I_to-encoding genes in hypertrophy, I_to was selectively reduced by overexpressing mutant dominant-negative (DN) transgenes. I_to amplitude was reduced equally (by about 50%) by overexpression of DN K_v1.4 (K_v1.4N) or DN K_v4.2 (either K_v4.2N or K_v4.2W362F), but only DN K_v4.2 prolonged APD duration (at 1 Hz) and induced myocyte hypertrophy. This hypertrophy was prevented by coexpressing wild-type K_v4.2 channels (K_v4.2F) with the DN K_v4.2 genes, suggesting the hypertrophy is due to I_to reduction and not nonspecific effects of transgene overexpression. The hypertrophy caused by reductions of K_v4.x-based I_to 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 K_v4.2N infection were prevented by blocking Ca²⁺ entry and excitability with verapamil or high [K⁺]_o. Our studies suggest that reductions of K_v4.2/3-based I_to play a role in hypertrophy signaling by activation of calcineurin.

Key Words: cardiac myocyte • [Ca²⁺]_i • transient outward K⁺ current • hypertrophy

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 (I_to) have consistently been observed in heart disease regardless of species.² In mammalian hearts, I_to has been shown to be encoded by K_v1.4, K_v4.2, and K_v4.3 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 K_v4.2 and K_v4.3 genes and associated changes in both I_to density and AP profile are commonly observed in myocytes from many animal models of heart disease^2,5,6 and human heart failure.⁷ Moreover, the magnitude of I_to and the level of expression of K_v4.2 and K_v4.3 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 K_v4.2/3 expression and heart disease, the link between reductions in I_to, K_v4.2/3 expression, and cardiac hypertrophy is still unclear, although reductions in I_to density and K_v4.2/3 expression occur very early after myocardial infarction in rats.⁶ APD prolongation following I_to reduction can increase Ca²⁺ influx through voltage-dependent L-type Ca²⁺ channels (I_Ca,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 I_to reduction might modulate hypertrophy signaling in myocardium. One particularly attractive candidate pathway, linking reductions in I_to 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 I_to reduction and hypertrophy was investigated in cultured neonatal rat ventricular myocytes. Using DN overexpression methods, reductions in K_v4.2/3-based I_to, but not K_v1.4-based I_to, causes myocyte hypertrophy that appears to require calcineurin activation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

Virus Constructs
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 K_v4.2 and K_v1.4 genes or the DN K_v4.2N (amino acids 1 to 311), K_v4.2M (K_v4.2W362F) (kindly provided by Dr J. Nerbonne, Washington University, St. Louis, Mo), and K_v1.4N (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).

Electrophysiological Recordings
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 2x10⁵ 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 Activity
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.

Statistics
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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Blockade of I_to Using Pharmacological Agents
To examine the consequences of long-term I_to reduction on cellular hypertrophy, we used Heteropoda toxin-3 (HpTx₃), a peptide spider toxin that blocks I_to 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) I_to 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) K_v4.2 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 I_to reduction by HpTx₃ induces hypertrophy as well as decreasing expression of I_to-encoding K_v4.2 protein.

View larger version (32K): [in this window] [in a new window]   Figure 1. Cell membrane capacitance (n=10) (A) and normalized 3H-leucine incorporation (n=5) (B) in cultured NRVM chronically treated with 400 nmol/L Heteropoda toxin-3 (HpTx3). Columns represent mean±SEM; *P<0.05 compared with GFP.

Electrophysiological Effects of DN K⁺ Channel Transgene Overexpression
Because cardiac I_to is encoded by K_v1.4, K_v4.2, and K_v4.3 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 I_to and myocyte hypertrophy. K_v1.4N overexpression was used to selectively reduce the portion of I_to encoded by members of the K_v1.x gene family, which have been directly linked to the slow component of I_to (I_to,s).^23–25 K_v4.2/3-based I_to, which underlies the fast component of I_to (I_to,f),^4,24 was reduced using two DN K_v4.2 transgenes, a truncated K_v4.2 gene (K_v4.2N), and a pore mutant (K_v4.2W362F or K_v4.2M), as used previously in transgenic mice models.^18,26 Figure 2A shows typical I_to traces recorded from GFP-, K_v1.4N-, K_v4.2N-, or K_v4.2M-infected myocytes in response to voltage steps to +60 mV from a holding potential of -90 mV. The corresponding average I_to amplitudes (per cell) are summarized in Figure 2B. This figure shows that overexpression of K_v1.4N decreased (P<0.001) I_to 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, I_to,s.²⁷ Similar reductions (P<0.001) were observed with overexpression of K_v4.2N (85.5±10.4 pA or 3.3±0.4 pA/pF, n=15) or K_v4.2M (80.9±7.4 pA or 3.2±0.3 pA/pF, n=22), but these constructs abolished the fast-recovering component, I_to,f.²⁷ The relative reductions of I_to by K_v1.4N versus K_v4.2N and K_v4.2M matches closely the relative proportions of I_to,s versus I_to,f that exist in NRVM.^15,27

View larger version (16K): [in this window] [in a new window]   Figure 2. A, Typical Ito traces recorded at +60 mV in cultured myocytes infected with adenoviruses expressing GFP, Kv1.4N, Kv4.2N, or Kv4.2M (left to right), and corresponding mean Ito amplitudes (B) (n=32, 28, 15, and 15, respectively). C, Typical action potential recordings at 1-Hz stimulation frequency for the same groups shown in (A). D, Mean time (ms) to 50% repolarization (APD50) (white column) and to 90% repolarization (APD90) (black column) at 1-Hz stimulation rate (n=11 for all groups). Columns represent mean±SEM; *P<0.05 compared with GFP.

Because I_to is an important current for the early repolarization of rat cardiac action potentials (APs), we anticipated that reductions of I_to would cause prolongation of APD. Typical APs recorded at 1 Hz stimulation rate from GFP-, K_v1.4N-, K_v4.2N-, and K_v4.2M-infected myocytes are presented in Figure 2C with Figure 2D summarizing averaged APD₅₀ and APD₉₀. Despite causing significant reductions in I_to, overexpression of K_v1.4N 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 K_v4.2N or K_v4.2M 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 K_v4.2N- and K_v4.2M-, but not in K_v1.4N-infected myocytes establishes that K_v1.4-based I_to,s makes minor contributions to APD¹⁵ as expected from the slow kinetics of recovery from inactivation of I_to,s (_rec,s=3 to 4 s) compared with K_v4.2/3-based I_to (_rec,f=80 to 100 ms).^4,28

Selective Effects of DN Channel Expression on Myocyte Hypertrophy
In addition to alterations in I_to current magnitude and APD, myocytes infected with K_v4.2N or K_v4.2M were noticeably larger than myocytes infected with either GFP or K_v1.4N when assessed using confocal microscopy (Figure 3A). Figure 3B shows that the averaged cell membrane capacitance in K_v4.2N- and K_v4.2M-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 K_v1.4N- (17.1±2.7 pF, n=31) infected myocytes. Moreover, Figure 3C shows that ³H-Leucine incorporation was also increased (P<0.001) following K_v4.2N (by 39.3±5.6%, n=15) or K_v4.2M (by 37.9±4.9%, n=22) infection compared with GFP- or K_v1.4N-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 K_v1.4 or K_v4.2 (K_v1.4F, K_v4.2F) 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 K_v4.2N- and K_v4.2M-infected myocytes is related to changes in I_to levels and APD.

View larger version (72K): [in this window] [in a new window]   Figure 3. A, Confocal images of myocytes infected with adenoviruses expressing GFP, Kv1.4N, Kv4.2N, or Kv4.2M. Infected myocytes appear green when excited at 480 nm and viewed through a 510-nm band-pass filter due to the presence of GFP. Cell capacitance (B) and normalized 3H-leucine incorporation (C) in the different groups. D, Actin filaments visualized by staining with Rhodamine-conjugated phalloidin in Kv4.2N compared with GFP-infected myocytes. Columns are mean±SEM; *P<0.05 compared with GFP.

Because contractile activity can have major effects on cell growth and hypertrophy signaling pathways,^29,30 it is conceivable that I_to 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 K_v4.2N- and K_v4.2M-infected myocytes). Moreover, overexpression of DN K_v4.2 in myocytes electrically paced at 2 Hz (for 48 hours) also enhanced ³H-leucine uptake (by 40.3±5.3% for K_v4.2N and by 31.5±6.2% for K_v4.2M) 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 K_v4.2 Using K_v4.2F
To explore further whether hypertrophy induced by K_v4.2N and K_v4.2M infection was related to reductions in I_to, DN K_v4.2-infected NRVMs were coinfected with K_v4.2F. As expected, K_v4.2F coexpression increased (P<0.001) I_to amplitude (1453.4±302.4 pA, n=15 for K_v4.2N+K_v4.2F and 1129.5±271.5 pA, n=13 for K_v4.2M+K_v4.2F) and abbreviated APD (APD₅₀=2.2±0.4 ms, APD₉₀=10.8±3.2 ms, n=18 for K_v4.2N+K_v4.2F and APD₅₀=2.8±0.8 and 12.4±2.1 ms, n=15 for K_v4.2M+K_v4.2F) compared with GFP, K_v4.2N or K_v4.2M infection alone. More importantly, K_v4.2F coinfection prevented increases in cell capacitance (18.9±1.0 pF, n=18 for K_v4.2N and 18.1±0.9 pF, n=15 for K_v4.2M) and ³H-leucine uptake (91.8±7.0% for K_v4.2N and 97.1±6.1% for K_v4.2M) compared with GFP-infected myocytes (P>0.4). As expected, overexpression of K_v4.2F alone elevated (P<0.001) I_to 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 K_v4.2N or K_v4.2M infection requires APD prolongation following reductions in I_to.

Hypertrophy Pathway: Calcineurin
We investigated the possible role of calcineurin in DN K_v4.2-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 K_v4.2N- and by 66.4±10.4% in K_v4.2M-infected myocytes (n=19) (Figure 4). These increases are comparable to the 63.5±5.9% increase in calcineurin phosphatase enzymatic activity following K_v4.2N 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 K_v4.2N-infected NRVM compared with GFP, without altering the degree of I_to reduction (81.7±17.2 pA, n=14) (Figure 5A). Overexpression of CAIN plus GFP did not measurably alter I_to 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 I_to reduction is dependent on Ca²⁺-activated calcineurin.

View larger version (14K): [in this window] [in a new window]   Figure 4. NFAT activity (a measure of calcineurin activity) was determined using a Dual Luciferase Reporter (DLR) assay in cultured myocytes infected with adenoviruses expressing GFP (n=19), GFP+CAIN (a specific inhibitor of calcineurin) (n=12), Kv4.2N (n=19), Kv4.2N+CAIN (n=12), Kv4.2M (n=19), or Kv4.2M+CAIN (n=12). Columns are mean±SEM; *P<0.05 compared with GFP.

View larger version (33K): [in this window] [in a new window]   Figure 5. Average effects of GFP, CAIN, Kv4.2N, and Kv4.2N+CAIN (n=14) on Ito amplitude (A) and cell membrane capacitance (B). C, The effects of DN Kv4.2 overexpression and coexpression with CAIN on cell 3H-leucine uptake. D, Typical images of myocytes infected with GFP, CAIN, Kv4.2N, and Kv4.2N+CAIN. Columns are mean±SEM; *P<0.05 compared with GFP.

Blockade of Myocyte Excitability and Inhibition of Ca²⁺ Influx
If calcineurin activation and induction of hypertrophy by DN K_v4.2 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 K_v4.2N (Figure 6). Treatment with verapamil reduced ³H-leucine uptake in K_v4.2N-infected myocytes by 41.6±6.9% (n=6) relative to untreated K_v4.2N-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 K_v4.2N-infected myocytes by 36.1±2.3% (n=4) relative to untreated K_v4.2N-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 I_to and APD prolongation after DN K_v4.2 overexpression. These observations demonstrate that cellular hypertrophy induced by DN K_v4.2 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 K_v4.2N-infected NRVM by verapamil and elevated [K⁺]_o (data not shown).

View larger version (54K): [in this window] [in a new window]   Figure 6. A, Typical confocal images of myocytes infected with Kv4.2N in the presence and absence of 10 µmol/L verapamil (blocker of ICa,L) or 50 mmol/L [K+]o (blocker of myocyte excitability). B, Mean 3H-leucine incorporation in GFP- or Kv4.2N-infected myocytes before and after treatment with 10 µmol/L verapamil or 50 mmol/L [K]o. Columns are mean±SEM; *P<0.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Different models of cardiac disease in mammals and humans are associated with prolonged action potential duration and reduced I_to.^2,7 Previous studies have established that K_v1.4, K_v4.2, and K_v4.3 channels encode for I_to currents^3,4,34 and are expressed in mammalian myocardium, although the relative contribution of these genes to I_to varies among different species in a developmental and region-specific fashion.^24,35–37 In addition, K_v1.7 channels also encode for I_to currents and are expressed in mammalian heart,²³ although their contribution to cardiac I_to is unclear. In heart disease and hypertrophy, the reductions in I_to are associated with decreased K_v4.2 and K_v4.3 expression.^5,6,9 The primary purpose of this study was to explore the possible contribution of reductions in I_to and K_v4.2/3 expression in triggering hypertrophy in cardiac myocytes.

Adenoviral infection of cultured NRVM with DN K_v4.2 and DN K_v1.4 transgenes were used to selectively eliminate currents generated by I_to-encoding cardiac genes.⁷ Taking advantage of the subfamily-specific nature of voltage-gated K⁺ channel assembly,³⁸ I_to currents generated by K_v1.4 (and possibly K_v1.7) were reduced using a truncated K_v1.4 transgene (K_v1.4N) and those generated by K_V4.2/3 channel genes were reduced using truncated (K_v4.2N) or pore-mutant (K_v4.2W362F or K_v4.2M) K_v4.2 transgenes.^18,26 K_v1.4N overexpression in cultured NRVM reduced I_to by about 50%, abolishing selectively slow I_to (I_to,s).²⁷ Overexpression of K_v4.2N or K_v4.2M also reduced I_to by about 50% but selectively eliminated the fast component of I_to (I_to,f) in NRVM.²⁷ Despite the similar reductions in I_to, only K_v4.2N/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 I_to,s (ie, _slow=2 to 3 s) limits its contribution to membrane repolarization at 1 Hz as reported previously.^15,27,39 By contrast, K_v4.2/3 channels recover rapidly from inactivation (_fast=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 K_v4.2/3-Based I_to
Our studies show that chronic treatment of cultured NRVM with HpTx3, a blocker of K_v4.2 and K_v4.3 channels,²¹ caused myocyte hypertrophy. An interesting feature of this hypertrophy was that the endogenous I_to,f was reduced in conjunction with downregulation of K_v4.2 expression as well as APD prolongation. These results suggest that reductions in I_to and APD prolongation can induce hypertrophy in cardiac myocytes as well as reduce the expression of the K_v4.2/3-encoding I_to genes, leading to a potential positive feedback.

Consistent with our findings with HpTx₃, overexpression of K_v4.2N or K_v4.2M 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 K_v4.2 strategies in NRVM was somewhat surprising because transgenic (TG) mice overexpressing the K_v4.2N transgene develop cardiac hypertrophy as well as enhanced contractility¹⁸ while mice overexpressing K_v4.2M are normal.²⁶ The lack of differences in the response of NRVM to K_v4.2N versus K_v4.2M overexpression seems inconsistent with previous suggestions that differences in the phenotypes between the corresponding TG models results from toxic effects of the K_v4.2N transgene.⁴⁰ Regardless, this hypertrophy induced by DN K_v4.2 (or HpTx₃) was not due to effects of these interventions on beating rate because spontaneous beating rates were not different between K_v1.4N-, K_v4.2N-, K_v4.2M-, and GFP-infected myocytes (see Mechanism of Hypertrophy section). Furthermore, DN K_v4.2 infection also induced hypertrophy in NRVM electrically stimulated at 2 Hz. By contrast, despite reducing I_to densities by 50%, DN K_v1.4 infection did not induce cardiac hypertrophy in spontaneously beating cultured NRVMs (at about 0.6 Hz). The presence of cardiac hypertrophy with DN K_v4.2 infection, combined with the absence of hypertrophy when K_v1.4-based currents were reduced, suggest that it is APD prolongation that induces hypertrophy in beating NRVMs with reduced I_to. Indeed, elimination of I_to,s by K_v1.4N is expected to only reduce total I_to amplitude by 10% to 20% when cells are beating spontaneously versus more than 80% reduction in I_to amplitude by DN K_v4.2 infection. These observations are consistent with the results from several rodent models of cardiac disease showing that K_v4.2 expression is reduced while K_v1.4 expression is increased^2,18 and results in NRVM treated with phenylephrine (an -adrenoreceptor agonist).¹⁰

Mechanism of Hypertrophy
We have demonstrated previously that reductions in K_v4.2/3-based I_to 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 ( and ß), 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 K_v4.2-infected myocytes for several reasons. First, blocking excitability and Ca²⁺ entry with verapamil or elevated [K⁺]_o prevented the hypertrophy induced by DN K_v4.2 overexpression without averting the reductions of I_to in cultured NRVM. Moreover, ³H-leucine incorporation following blockade of excitability was normalized between GFP- and DN K_v4.2-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 I_to reduction was further supported by the ability of K_v4.2F coinfection to elevate I_to, shorten APD, and prevent myocyte hypertrophy induced by DN K_v4.2, as well as the lack of hypertrophy in NRVM overexpressing K_v1.4N (which does not prolong APD). Third, overexpression of DN K_v4.2 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 K_v4.2N construct.³³

Although numerous cell signaling pathways might be involved in transduction of I_to 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 K_v4.2N or K_v4.2M. 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 K_v4.2 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 K_v4.2N,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 K_v4.2N or K_v4.2M along with full length K_v4.2F, the number of PFUs per cell was increased (15 to 20 PFU/cell) and produced no evidence of cell death. More importantly, coinfection with K_v4.2F elevated I_to, shortened APD, and prevented the myocyte hypertrophy induced by DN K_v4.2. Although I_to was increased well above the control levels following K_v4.2F coinfection, attempt to reduce the I_to 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 K_v4.2F 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 I_to produced by K_v4.2/3 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.

	Acknowledgments

 
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 K_v4.2W362F cDNA. We also acknowledge NPS Pharmaceuticals for the kind gift of HpTx₃.

Received August 24, 2001; revision received January 18, 2002; accepted January 22, 2002.

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