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(Circulation. 2002;105:1850.)
© 2002 American Heart Association, Inc.

Basic Science Reports

Inhibition of Calcineurin and Sarcolemmal Ca²⁺ Influx Protects Cardiac Morphology and Ventricular Function in K_v4.2N Transgenic Mice

Rajan Sah, PhD; Gavin Y. Oudit, MSc, MD; The-Tin T. Nguyen, MSc; Hae W. Lim, PhD; Alan D. Wickenden, PhD; Gregory J. Wilson, MD; Jeffery D. Molkentin, PhD; Peter H. Backx, DVM, PhD

From the Departments of Physiology and Medicine and the Division of Cardiology, University Health Network, University of Toronto, Toronto, Canada (R.S., G.Y.O., T.-T.T.N., P.H.B.); the Department of Pediatrics, University of Cincinnati, Children’s Hospital Medical Center, Cincinnati, Ohio (H.W.L., J.D.M.); ICAgen Inc, Durham, NC (A.D.W.); and the Divisions of Cardiovascular Research and Pathology, Hospital for Sick Children, Toronto, and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada (G.J.W.).

Correspondence to Peter H. Backx, DVM, PhD, Heart and Stroke/Richard Lewar Centre, Fitzgerald Building, Rm 68, 150 College St, Toronto, Ontario M55 3EZ, Canada. E-mail p.backx{at}utoronto.ca

	Abstract

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Background— Cardiac-targeted expression of truncated K_v4.2 subunit (K_v4.2N) reduces transient outward current (I_to) density, prolongs action potentials (APs), and enhances contractility in 3- to 4-week-old transgenic mice. By 13 to 15 weeks of age, these mice develop severely impaired cardiac function and signs of heart failure. In this study, we examined whether augmented contractility in K_v4.2N mice results from elevations in intracellular calcium ([Ca²⁺]_i) secondary to AP prolongation and investigated the putative roles of calcineurin activation in heart disease development of K_v4.2N mice.

Methods and Results— At 3 to 4 weeks of age, L-type Ca²⁺ influx and peak [Ca²⁺]_i were significantly elevated in K_v4.2N myocytes compared with control because of AP prolongation. Cardiac calcineurin activity was also significantly elevated in K_v4.2N mice by 5 weeks of age relative to controls and increased progressively as heart disease developed. This was associated with activation of protein kinase C (PKC)- and PKC- but not PKC-, as well as increases in ß-myosin heavy chain (ß-MHC) and reductions in sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA)-2a expression. Treatment with either cyclosporin A or verapamil prevented increases in heart weight to body weight ratios, interstitial fibrosis, impaired contractility, PKC activation, and changes in the expression patterns of ß-MHC and SERCA2a.

Conclusions— Our results demonstrate that AP prolongation caused by I_to reduction results in enhanced Ca²⁺ cycling and hypercontractility in mice and suggests that elevations in [Ca²⁺]_i via I_Ca,L and activation of calcineurin play a central role in disease development after I_to reduction using the K_v4.2N construct.

Key Words: action potentials • contractility • cardiomyopathy • ion channels

	Introduction

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The cardiac Ca²⁺-independent transient outward current (I_to) is encoded by K_v4.2, K_v4.3, and K_v1.4 genes.¹ Reduction in I_to, as typically occurs in heart disease, results in prolongation of the early phase of the cardiac action potential (AP).² Electrophysiological consequences of increased AP duration in heart disease include QT prolongation³ and increased arrhythmias.⁴ Indeed, reduced K⁺ currents in transgenic mice can cause AP prolongation,^5–7 lengthened QT intervals, and ventricular tachyarrhythmias.^5,7

AP prolongation as a result of I_to reduction in rodent hearts also elevates intracellular Ca²⁺ transients via increased L-type Ca²⁺ influx (I_Ca,L),^8,9 thereby leading to enhanced myocardial contractility. The finding that I_to downregulation and associated AP prolongation occur early in the disease process¹⁰ suggests that this may represent a compensatory mechanism to enhance contractility of the compromised myocardium.¹¹

In addition to enhancing contractility, increased Ca²⁺ entry via I_Ca,L after AP prolongation might also contribute to the myocyte hypertrophy commonly seen in heart disease by acting as a stimulus for cellular growth through the activation of cell-signaling pathways,¹¹ such as the calcineurin-dependent pathway¹² or the interconnected protein kinase C (PKC)-–dependent pathway.^13,14 Recently, this Ca²⁺ hypothesis was directly supported in mice overexpressing the L-type Ca²⁺ channel in the heart, in which a sustained increase in I_Ca,L resulted in severe cardiomyopathy by 8 months of age via PKC- activation.¹³

Previously, we showed that overexpressing an N-terminal fragment of K_v4.2 (K_v4.2N) reduced I_to and prolonged APs in transgenic mice in a dominant-negative manner.⁶ The hearts of K_v4.2N mice were hypercontractile at 2 to 4 weeks of age and progressively developed cardiac hypertrophy with chamber dilatation by 13 to 15 weeks of age. In this study, we demonstrate that reductions in I_to and resultant AP prolongation enhance both transsarcolemmal Ca²⁺ influx and intracellular Ca²⁺ in K_v4.2N myocytes. Furthermore, the transition from hypercontractility to cardiomyopathy in K_v4.2N hearts can be prevented by either Ca²⁺ channel blockade or calcineurin inhibition, supporting the notion that aberrant Ca²⁺ cycling leading to activation of calcineurin may contribute to the initiation of heart disease.

	Methods

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K_v4.2N Transgenic Mice
Transgenic mice overexpressing a truncated K_v4.2 subunit (K_v4.2N) were generated as reported previously.⁶

Cardiomyocyte Isolation
Mouse ventricular myocytes were isolated from 2- to 4-week-old control and transgenic mice as previously described.⁶

Electrophysiology and Intracellular Ca²⁺ Measurements
Electrophysiological and intracellular Ca²⁺ recordings were performed using the whole-cell patch-clamp technique as previously described.⁹ For AP and intracellular Ca²⁺ transient measurements, the extracellular solution was composed of (mmol/L): 140 NaCl, 4 KCl, 10 HEPES, 1 MgCl₂, 2 CaCl₂, and 10 D-glucose, adjusted to pH 7.4 with NaOH. For I_Ca,L measurements, the extracellular solution contained (mmol/L): 135 choline-Cl, 10 HEPES, 1 MgCl₂, 2 CaCl₂, and 10 D-glucose, adjusted to pH 7.4 with CsOH. The pipette solution for AP measurements contained (mmol/L): 140 KCl, 10 HEPES, 1 MgCl₂, 10 NaCl, 7 Mg-ATP, and 0.1 BAPTA, adjusted to pH 7.2 with KOH. The same internal solution was used for Ca²⁺ measurements, but 100 µmol/L BAPTA was replaced with 60 µmol/L fura 2 pentapotassium salt. Pipette solutions for I_Ca,L contained (mmol/L): 120 aspartic acid, 120 CsOH, 10 HEPES, 1 MgCl₂, 7 Mg-ATP, 10 mmol/L TEA-Cl, and 10 EGTA, adjusted to pH 7.2 with CsOH. APs were evoked in current-clamp mode by a brief 5-ms current injection. Intracellular Ca²⁺ and I_Ca,L were measured with current-clamp and AP-clamp recordings after steady-state stimulation at a frequency of 0.25 Hz.

Calcineurin Activity
Calcineurin phosphatase enzymatic activity was measured in duplicate from cardiac protein extracts obtained from 3 to 5 hearts as described previously.¹⁵

Treatment Protocol
Male transgenic K_v4.2N mice and littermate controls were divided into 4 groups at 2 to 3 weeks of age for either cyclosporin A (CsA) or verapamil treatment: control+vehicle, control+CsA (or verapamil), K_v4.2N+vehicle, and K_v4.2N+CsA (or verapamil). The vehicle used for CsA was the castor oil–based solvent cremaphor (Sigma), and for verapamil, 3% dextrose was used. CsA-treated mice were injected once daily subcutaneously with a dose of 30 mg/kg of CsA (Calbiochem), whereas vehicle-treated mice received an equivalent volume of cremaphor. Verapamil (Sigma) was administered through drinking water (0.025 mg/mL). CsA was withdrawn for 48 hours, and verapamil was withdrawn for 24 hours before assessment of ventricular function. Direct effects of CsA on contractile function were assessed in control mice after 4 weeks of treatment without drug withdrawal.

Histology
After each mouse was euthanized, hearts were removed and either snap-frozen in liquid nitrogen for Western analysis or calcineurin assays or fixed in 10% buffered formalin for histopathology. At least 3 hearts from each group were bisected midway between base and apex and embedded in paraffin. Thin sections (5 µmol/L) were cut on a rotary microtome and stained with hematoxylin/eosin or elastic trichrome.

Hemodynamic Indexes
Mice were anesthetized with ketamine (50 to 100 mg/kg IP) and xylazine (3 to 6 mg/kg IP). The right common carotid was cannulated with a 1.4F Millar catheter (Millar Inc), which was advanced into the proximal aorta and into the left ventricular cavity. Pressure recordings were filtered at 300 Hz and sampled at 2 kHz (2801A, Data Translation). The time constant for isovolumic relaxation, tau (), was determined by fitting the bottom half of the left ventricular pressure traces with a monoexponential function, P(t)=P_oe^-t/.

Echocardiography
After anesthesia (see above), transthoracic 2D, M-mode, and Doppler echocardiographic examination was performed with an Acuson Sequoia C256 system equipped with a 15-MHz linear transducer (15L8) (version 4.0, Acuson Corp).

Western Analysis and PKC Translocation
Protein (10 µg) from the total homogenates of individual mouse hearts were analyzed by Western blot as previously described¹⁶ with monoclonal antibodies to sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA)-2a (Santa Cruz Biotechnology), ß-myosin heavy chain (ß-MHC) (kindly provided by Dr Jeffery Robbins, Cincinnati, Ohio), and calsequestrin (Santa Cruz Biotechnology), which was used as an internal standard. Membrane and cytosolic fractions of heart homogenates were isolated as previously described.¹⁴ Proteins were separated on an 8% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were stained with Ponceau S to check for equal protein loading and then cut into 2 halves: the upper half was probed with PKC-, PKC-, or PKC- antibodies, and the bottom half was probed with calsequestrin antibody (Santa Cruz Biotechnology), which was used as an internal standard.

Statistical Analysis
A 1-way ANOVA was used to test for overall significance, followed by the Student-Newman-Keuls test for multiple comparison testing between the various groups. The unpaired t test was used to compare the means of 2 groups. All statistical analyses were performed with the SPSS software (version 10.1). A value of P<0.05 was considered statistically significant. Data are presented as mean±SEM; n refers to the sample size.

	Results

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Effects of K_v4.2N Overexpression on AP Duration, Ca²⁺ Influx, and [Ca²⁺]_i Transients in 3- to 5-Week-Old Mice
As reported previously,⁶ overexpressing a dominant-negative truncated mutant K_v4.2 subunit prolongs AP duration (APD; Figure 1A) in K_v4.2N myocytes (APD_50,Kv4.2N=9.9±0.7 ms, APD_90,Kv4.2N=35.2±5.1 ms; n=4) compared with controls (APD_50,CON=3.1±0.4 ms, APD_90,CON=20.2±3.6 ms; n=8).⁶ The increased APD₅₀ in K_v4.2N myocytes is consistent with the dominant contribution of I_to,f to repolarization in mouse myocardium.¹⁷ As expected from previous reports,^9,18 total Ca²⁺ entry through L-type Ca²⁺ channels (Q_Ca,L) was elevated (P<0.01) nearly 3-fold in transgenic myocytes (Q_Ca,L=14.8±1.7 picocoulombs, n=6) stimulated with typical K_v4.2N APs compared with control myocytes (Q_Ca,L=5.1±0.9 picocoulombs, n=7) driven by control APs (Figure 1B). Despite these differences in integrated Ca²⁺ entry per beat, I_Ca,L densities evoked by voltage steps to +10 mV did not differ significantly between control (15.9±1.9 pA/pF, n=6) and K_v4.2N (15.2±1.5 pA/pF, n=6) myocytes. These results demonstrate that I_Ca,L is highly dependent on AP profile in mouse cardiac myocytes and that AP prolongation in K_v4.2N mice produces large increases in Q_Ca,L, independent of changes in L-type Ca²⁺ channel densities.

View larger version (18K): [in this window] [in a new window]   Figure 1. AP records, L-type Ca2+ currents, and intracellular [Ca2+]i transients in control and Kv4.2N mice. A, APs recorded from Kv4.2N myocytes are lengthened relative to control. B, Calcium currents measured in control (left) and Kv4.2N (right) myocytes under AP-clamp conditions using the APs shown in A. C, [Ca2+]i transients measured in current-clamp mode from control and Kv4.2N myocytes driven under their own short and long APs, respectively. D, Peak [Ca2+]i measured in AP-clamp mode from a control myocyte stimulated with a long Kv4.2N AP (left) and a Kv4.2N myocyte stimulated with a short control AP (right).

To determine whether intracellular Ca²⁺ is also altered in K_v4.2N myocytes, [Ca²⁺]_i transients were recorded in myocytes isolated from 3- to 5-week-old mice (Figure 1C). Under current-clamp conditions, intracellular [Ca²⁺]_i transients were increased (P<0.05) in K_v4.2N myocytes (583±115 nmol/L, n=3) compared with control myocytes (308±31 nmol/L, n=6) (Figure 1C). Despite these differences in peak systolic Ca²⁺, no differences were observed in diastolic [Ca²⁺]_i between K_v4.2N and control myocytes at the early time point. Furthermore, stimulation of K_v4.2N myocytes with control APs normalized peak systolic intracellular Ca²⁺ (315±111 nmol/L, n=3) and, conversely, control myocytes stimulated with typical K_v4.2N APs yielded elevated Ca²⁺ transients (608±55 nmol/L, n=6), indistinguishable (P=0.82) from K_v4.2N myocytes under current-clamp conditions (Figure 1D).

Role of Calcineurin in K_v4.2N Mice
The above results suggest that cardiac hypercontractility in early-stage K_v4.2N hearts results from AP prolongation and increased Ca²⁺ influx through I_Ca,L. These sustained elevations in [Ca²⁺]_i and I_Ca,L might also contribute to the transition from hyperfunction to heart failure in the K_v4.2N mice via activation of Ca²⁺-dependent signaling pathways.¹¹ Recently, the Ca²⁺-activated phosphatase calcineurin has been identified as an important signaling protein for the induction of hypertrophy in numerous animal models^19,20 as well as in humans.²¹ Therefore, calcineurin activity was measured in both control and K_v4.2N mice before (5 to 8 weeks of age) and after (13 to 15 weeks of age) the development of overt heart disease in the K_v4.2N mice. Figure 2, A through C, shows that the calcineurin activity is elevated in K_v4.2N hearts, precedes disease development, and increases progressively (P<0.05) with age. To establish a causal relationship between calcineurin activation and disease initiation, K_v4.2N mice were treated with the calcineurin inhibitor CsA for 12 weeks beginning at 3 weeks of age. Figure 3A shows that calcineurin inhibition effectively reduced (P<0.05) the ratio of heart weight (HW) to body weight (BW) of K_v4.2N mice (HW/BW_Kv4.2N,Veh=0.57±0.06, n=10; HW/BW_Kv4.2N,CsA=0.44±0.02, n=8) to control levels. Furthermore, treatment with CsA did not affect body weight in either control or K_v4.2N mice (data not shown).

View larger version (26K): [in this window] [in a new window]   Figure 2. Calcineurin activity in Kv4.2N mice throughout development. Calcineurin activity measured in Kv4.2N mice at 5, 8, and 13 weeks of age was elevated 1.53-fold, 1.85-fold, and 2.92-fold, respectively, relative to littermate controls. *P<0.05 between control and Kv4.2N, P<0.001 between 13-week-old and 5- and 8-week-old Kv4.2N mice.

View larger version (40K): [in this window] [in a new window]   Figure 3. HW/BW ratios after CsA (A) and verapamil (B) treatment. Neither CsA nor verapamil changed HW/BW ratios of control mice, whereas both were effective at normalizing HW/BW ratios of Kv4.2N mice. *P<0.05 between untreated Kv4.2N mice and all other groups.

Trichrome staining shows that extensive cardiomyocyte vacuolization and interstitial fibrosis accompany myocyte hypertrophy as assessed by cell-membrane capacitance (232±16 pF; n=16) in vehicle-treated 15-week-old K_v4.2N hearts (Figure 4B) compared with vehicle-treated controls (156±6 pF; n=23; Figure 4A). The causes of the excessive collagen deposition in late-stage K_v4.2N hearts are unclear but are consistent with myocyte dropout,⁶ potentially arising from calcineurin-dependent apoptosis.²² By contrast, CsA treatment of K_v4.2N mice markedly reduced these pathological changes (Figure 4C).

View larger version (138K): [in this window] [in a new window]   Figure 4. Elastic trichrome–stained histological sections (x400) of hearts from control and treated/untreated Kv4.2N mice. At 15 weeks of age, vehicle-treated control hearts were normal histologically (A), whereas Kv4.2N hearts (B) showed extensive cardiomyocyte vacuolization and interstitial collagen deposition (green). Both CsA (C) and verapamil (D) substantially reduced the amount of cardiomyocyte vacuolization and interstitial fibrosis compared with the vehicle-treated late-stage Kv4.2N hearts (B).

Functional characterization of hearts from 13- to 15-week-old vehicle-treated and CsA-treated K_v4.2N mice showed that systolic and diastolic ventricular function was significantly improved with CsA treatment. In fact, fractional shortening, heart-rate–corrected velocity of fractional shortening (Table 1), and +dP/dt (Table 2) were all enhanced (P<0.05) relative to control, as observed in untreated K_v4.2N mice before the onset of pathology. These findings did not appear to result from direct effects of CsA, because CsA treatment reduced contractility in 7-week-old control mice, consistent with previous findings,²³ whereas K_v4.2N treated with CsA mice at this age remained hypercontractile (data not shown). These results demonstrate that enhanced ventricular function in 13- to 15-week-old, CsA-treated K_v4.2N mice reflects a genuine hypercontractility, probably arising from AP prolongation. Although diastolic function was not enhanced in CsA-treated K_v4.2N mice, normalization of the passive versus active components of mitral flow (E/A ratios),²⁴ as well as restoration of -dP/dt and rate of ventricular pressure decay () (Tables 1 and 2), collectively suggest that normal diastolic function was preserved in CsA-treated K_v4.2N mice.

View this table: [in this window] [in a new window]   Table 1. Echocardiographic Characterization of 13- to 15-Week-Old Verapamil- and CsA-Treated Mice

View this table: [in this window] [in a new window]   Table 2. Hemodynamic Characterization of 13- to 15-Week-Old Verapamil- and CsA-Treated Mice

Effects of Verapamil Treatment on Cardiac Morphology and Ventricular Function
Having established that calcineurin activation is critical for disease progression in K_v4.2N mice, we next tested whether the heart disease process depended on I_Ca,L. Because the total integrated I_Ca,L (Q_Ca,L) is increased 3-fold as a result of AP prolongation in K_v4.2N myocytes and because the activity of numerous hypertrophic signaling pathways, including the calcineurin pathway, are Ca²⁺-dependent,²⁵ we examined whether treatment of K_v4.2N mice with L-type Ca²⁺ channel blockers could modify the course of the disease. As seen with CsA, verapamil treatment also normalized HW/BW ratios (HW/BW_Kv4.2N-Veh=0.51±0.03, n=6; HW/BW_Kv4.2N-Verap= 0.35±0.02, n=8; HW/BW_CON,Veh=0.38±0.01) (Figure 3B) and improved the cardiac morphology of K_v4.2N mice by reducing interstitial fibrosis and cardiomyocyte vacuolization (Figure 4D) compared with vehicle-treated K_v4.2N hearts (Figure 4B).

Both systolic and diastolic ventricular function as assessed by echocardiographic and hemodynamic measures (Tables 1 and 2) were also restored to control levels in verapamil-treated K_v4.2N mice but were not hypercontractile as observed with CsA treatment. This suggests that L-type Ca²⁺ channel inhibition at the doses in our studies could not protect K_v4.2N mice sufficiently against disease to preserve the hyperfunctional state.

Expression of SERCA2a and ß-MHC and PKC Activation
Reduced expression of SERCA2a and enhanced expression of ß-MHC are universal molecular markers of cardiac dysfunction and disease (Figure 5).^26,27 In untreated K_v4.2N mice at 13 to 15 weeks of age, cardiac SERCA2a protein levels were significantly (P<0.05) reduced (17.5±1.8 arbitrary units [AU], n=8) compared with control SERCA2a levels (38.7±2.1 AU, n=4), and this reduction was prevented after 10 to 13 weeks of treatment with either verapamil (39.9±1.4 AU, n=4) or CsA (36.9±2.0 AU, n=4) (Figure 5A and 5B). Similarly, ß-MHC protein expression was elevated 8.2-fold (P<0.05) in untreated 13- to 15-week-old K_v4.2N mice (4.38±0.70 AU, n=8) relative to controls (0.54±0.35 AU, n=4). This increase was significantly reduced (P<0.05) in both verapamil-treated (1.39±1.00 AU, n=4) and CsA-treated (0.98±0.63 AU, n=4) K_v4.2N mice to levels indistinguishable from control (P>0.05; Figure 5A and 5C).

View larger version (35K): [in this window] [in a new window]   Figure 5. Western blots of SERCA2a and ß-MHC protein. SERCA2a protein (A and B) is reduced by 55% and ß-MHC increased 8-fold (A and C) in 15-week-old vehicle-treated Kv4.2N hearts relative to vehicle-treated controls. Treatment of Kv4.2N mice with either CsA or verapamil completely restored the level of SERCA2a expression to normal and decreased ß-MHC expression to near control levels. Blots were probed with calsequestrin antibody to demonstrate equal protein loading. *P<0.05 between Kv4.2N and all other groups.

Because calcineurin activation is associated with a characteristic pattern of PKC isoform activation,¹⁴ the membrane translocation patterns of the PKC-, -, and - isoforms were examined. The membrane-to-cytosol ratios of the PKC- and PKC- isoforms were increased 6.1- and 2.4-fold, respectively, in K_v4.2N mice relative to controls (Figure 6, A through C). Consistent with a connection between PKC and calcineurin pathways, treatment of K_v4.2N mice with either verapamil or CsA normalized both PKC- and - expression patterns (Figure 6, A through C). In contrast, PKC- activation did not differ among any of the groups studied (Figure 6, A and D).

View larger version (68K): [in this window] [in a new window]   Figure 6. Activation profile of PKC. A, Western blot analysis of PKC-, PKC-, and PKC- in both cytosolic and membrane protein fractions. Each band reflects 4 pooled hearts, and the experiment was performed in duplicate or quadruplicate. Histogram representations of PKC- (B), - (C), and - (D) protein levels demonstrate that PKC- and - increase in the membrane fraction of Kv4.2N hearts, whereas total PKC- protein levels and distribution remains unchanged in all groups. Both CsA and verapamil restored the subcellular distribution of PKC- and - in Kv4.2N hearts.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Diminished I_to density and altered AP profile have been associated with heart disease in humans and in numerous animal models.^1,2,18,28 This I_to reduction occurs early in the disease process,¹⁰ suggesting that I_to may play a role in the initiation of disease.¹¹ Previously, we found that primary I_to reduction achieved by cardiac-restricted overexpression of a truncated K_v4.2N peptide in mice causes enhanced ventricular function that progresses to failure by 15 weeks of age.⁶ In this study, we investigated the potential role of Ca²⁺ in the enhanced contractility and progression of disease in K_v4.2N overexpressing mice.

Consistent with previous studies in rat myocytes,^8,9,18 AP prolongation in K_v4.2N transgenic myocytes increased peak [Ca²⁺]_i, supporting the notion that I_to plays an important role in influencing Ca²⁺ handling and contractility in situations, such as heart disease, in which I_to densities are reduced.^8,9,18 Our results are the first to demonstrate enhanced Ca²⁺ transients and inotropy via altered AP profile in transgenic mice with selective I_to reductions.

Associated with elevated intracellular Ca²⁺, calcineurin activity increased with disease progression, consistent with previous studies establishing a central role for calcineurin activation in animal models of cardiac hypertrophy^12,19,20 and during the hypertrophic phase of human heart disease.²¹ The finding that calcineurin activity was elevated in K_v4.2N mice before the onset of disease prompted us to attempt to prevent disease development by inhibiting I_Ca,L or calcineurin. The high degree of protection conferred to K_v4.2N transgenic mice by verapamil or CsA suggests that the transition from hyperfunction to failure in K_v4.2N mice is Ca²⁺-dependent.

Recently, Muth et al^13,29 showed that cardiac-specific overexpression of voltage-dependent L-type Ca²⁺ channels (L-VDCCs) resulted in increased Ca²⁺ influx and enhanced basal contractility in mice at 8 weeks of age²⁹ and progression to cardiomyopathy by 8 months of age.¹³ The striking similarity in activation of signaling pathways and overall phenotype between the L-VDCCs and the K_v4.2N mice suggests common mechanisms. Indeed, both transgenic models generate chronically elevated I_Ca,L (by 50% in L-VDCC mice¹³ and by 200% in K_v4.2N mice) and show activation of Ca²⁺-dependent PKC- but not PKC-. Although calcineurin signaling was not examined in L-VDCC mice,¹³ calcineurin activation has recently been associated with a characteristic activation profile of PKC isoforms (PKC- and - and not -, -ß, or -) and mitogen-activated protein kinase signaling pathways (JNK and not p38 or ERK1/2).¹⁴ The patterns of PKC isoform activation observed in our studies in association with calcineurin activation are consistent with these findings and suggest that the calcineurin pathway is the dominant hypertrophic signaling pathway in K_v4.2N mice.

Regardless of the specific hypertrophic signaling pathways involved, the phenotypes observed in both K_v4.2N and L-VDCC transgenic mice implicate Ca²⁺-dependent mechanisms.¹¹ These observations may be clinically relevant, because the initial stage of heart disease is typically characterized by I_to downregulation^2,10 and neurohumoral activation,³⁰ both of which may elevate Ca²⁺ influx and intracellular Ca²⁺, thereby helping to maintain adequate cardiac output from the diseased heart. Our results suggest that normalization of Ca²⁺ cycling via I_Ca,L blockade or decoupling of the beneficial inotropic effects of Ca²⁺ from its detrimental hypertrophic effects via inhibition of Ca²⁺-dependent signaling pathways may present a useful therapeutic strategy for heart disease treatment.

Interestingly, the absence of cardiac pathology in the phospholamban knockout mouse, which also exhibits elevated intracellular [Ca²⁺]_i via enhancements in SR Ca²⁺ uptake,³¹ suggests that the source of the Ca²⁺ imbalance (I_Ca,L versus SERCA2a:phospholamban ratio) might be an important factor in disease initiation. The subsarcolemmal colocalization of calcineurin³² and PKC with the L-type Ca²⁺ channel via the A-kinase anchoring protein AKAP79³³ supports the notion that these molecules may respond preferentially to local elevations in submembrane Ca²⁺, as expected after enhancements in I_Ca,L versus bulk cytosolic Ca²⁺.

The discrepancy in the phenotypes observed in the K_v4.2N and K_v4.2W362F mice,⁷ despite similar electrophysiological findings, suggests that secondary effects of K_v4.2N peptide expression,⁶ in addition to enhanced Ca²⁺ cycling, may contribute in part to the cardiomyopathy in K_v4.2N mice. Recent studies in our laboratory, however, have demonstrated that overexpression of either K_v4.2N or K_v4.2W362F transgenes in neonatal rat ventricular myocytes both result in similar levels of Ca²⁺-calcineurin–dependent hypertrophy.³⁴ Furthermore, consistent with our findings, additional studies have now revealed that pressure-overload–induced cardiac hypertrophy is enhanced in K_v4.2W362FxKv1.4^-/- mice relative to controls,³⁵ suggesting that I_to reduction and AP prolongation do, in fact, contribute to myocyte hypertrophy.

In summary, we have shown that AP prolongation as a result of I_to reduction by overexpression of the K_v4.2N peptide increases contractility in early-stage transgenic myocytes by increasing I_Ca,L and intracellular [Ca²⁺]_i. Progression to failure then occurs via a Ca²⁺- and calcineurin-dependent process that is associated with activation of PKC- and - but not - and can be inhibited by verapamil and CsA. Although a secondary, indirect effect of K_v4.2N expression may also contribute to disease in these hearts, our data clearly show that I_Ca,L, [Ca²⁺]_i, and calcineurin are dominant players in the pathology observed after AP prolongation in K_v4.2N hearts. Furthermore, these findings are consistent with a "Ca²⁺ hypothesis" of hypertrophic gene expression induced by sustained elevations in Ca²⁺ influx and/or intracellular [Ca²⁺]_i.

	Acknowledgments

 
This study was supported by a Canadian Institutes of Health Research (CIHR) grant to Dr Backx. Dr Sah holds an MD/PhD studentship from the CIHR, and Dr Oudit holds a postdoctoral fellowship from CIHR. Dr Backx is a Career Investigator of the Heart and Stroke Foundation of Ontario. We are grateful for equipment support from the Richard Lewar/Heart and Stroke Center of Excellence. We also thank Michael Lee-Poy and Roberto J. Diaz Jr for assistance in these studies.

Received November 14, 2001; revision received February 4, 2002; accepted February 5, 2002.

	References

Top Abstract Introduction Methods Results Discussion References

 

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