Inhibition of Calcineurin and Sarcolemmal Ca2+ Influx Protects Cardiac Morphology and Ventricular Function in Kv4.2N Transgenic Mice
Background— Cardiac-targeted expression of truncated Kv4.2 subunit (Kv4.2N) reduces transient outward current (Ito) 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 Kv4.2N 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 Kv4.2N mice.
Methods and Results— At 3 to 4 weeks of age, L-type Ca2+ influx and peak [Ca2+]i were significantly elevated in Kv4.2N myocytes compared with control because of AP prolongation. Cardiac calcineurin activity was also significantly elevated in Kv4.2N 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)-α and PKC-θ but not PKC-ε, as well as increases in β-myosin heavy chain (β-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 β-MHC and SERCA2a.
Conclusions— Our results demonstrate that AP prolongation caused by Ito reduction results in enhanced Ca2+ cycling and hypercontractility in mice and suggests that elevations in [Ca2+]i via ICa,L and activation of calcineurin play a central role in disease development after Ito reduction using the Kv4.2N construct.
Received November 14, 2001; revision received February 4, 2002; accepted February 5, 2002.
The cardiac Ca2+-independent transient outward current (Ito) is encoded by Kv4.2, Kv4.3, and Kv1.4 genes.1 Reduction in Ito, 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 Ito reduction in rodent hearts also elevates intracellular Ca2+ transients via increased L-type Ca2+ influx (ICa,L),8,9⇓ thereby leading to enhanced myocardial contractility. The finding that Ito 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 ICa,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)-α–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 ICa,L resulted in severe cardiomyopathy by 8 months of age via PKC-α activation.13
Previously, we showed that overexpressing an N-terminal fragment of Kv4.2 (Kv4.2N) reduced Ito and prolonged APs in transgenic mice in a dominant-negative manner.6 The hearts of Kv4.2N 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 Ito and resultant AP prolongation enhance both transsarcolemmal Ca2+ influx and intracellular Ca2+ in Kv4.2N myocytes. Furthermore, the transition from hypercontractility to cardiomyopathy in Kv4.2N 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.
Kv4.2N Transgenic Mice
Transgenic mice overexpressing a truncated Kv4.2 subunit (Kv4.2N) were generated as reported previously.6
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 ICa,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 ICa,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 ICa,L were measured with current-clamp and AP-clamp recordings after steady-state stimulation at a frequency of 0.25 Hz.
Calcineurin phosphatase enzymatic activity was measured in duplicate from cardiac protein extracts obtained from 3 to 5 hearts as described previously.15
Male transgenic Kv4.2N 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), Kv4.2N+vehicle, and Kv4.2N+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.
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.
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 (τ), was determined by fitting the bottom half of the left ventricular pressure traces with a monoexponential function, P(t)=Poe−t/τ.
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), β-myosin heavy chain (β-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-α, PKC-θ, or PKC-ε antibodies, and the bottom half was probed with calsequestrin antibody (Santa Cruz Biotechnology), which was used as an internal standard.
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.
Effects of Kv4.2N 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 Kv4.2 subunit prolongs AP duration (APD; Figure 1A) in Kv4.2N myocytes (APD50,Kv4.2N=9.9±0.7 ms, APD90,Kv4.2N=35.2±5.1 ms; n=4) compared with controls (APD50,CON=3.1±0.4 ms, APD90,CON=20.2±3.6 ms; n=8).6 The increased APD50 in Kv4.2N myocytes is consistent with the dominant contribution of Ito,f to repolarization in mouse myocardium.17 As expected from previous reports,9,18⇓ total Ca2+ entry through L-type Ca2+ channels (QCa,L) was elevated (P<0.01) nearly 3-fold in transgenic myocytes (QCa,L=14.8±1.7 picocoulombs, n=6) stimulated with typical Kv4.2N APs compared with control myocytes (QCa,L=5.1±0.9 picocoulombs, n=7) driven by control APs (Figure 1B). Despite these differences in integrated Ca2+ entry per beat, ICa,L densities evoked by voltage steps to +10 mV did not differ significantly between control (15.9±1.9 pA/pF, n=6) and Kv4.2N (15.2±1.5 pA/pF, n=6) myocytes. These results demonstrate that ICa,L is highly dependent on AP profile in mouse cardiac myocytes and that AP prolongation in Kv4.2N mice produces large increases in QCa,L, independent of changes in L-type Ca2+ channel densities.
To determine whether intracellular Ca2+ is also altered in Kv4.2N 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 Kv4.2N 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 Kv4.2N and control myocytes at the early time point. Furthermore, stimulation of Kv4.2N myocytes with control APs normalized peak systolic intracellular Ca2+ (315±111 nmol/L, n=3) and, conversely, control myocytes stimulated with typical Kv4.2N APs yielded elevated Ca2+ transients (608±55 nmol/L, n=6), indistinguishable (P=0.82) from Kv4.2N myocytes under current-clamp conditions (Figure 1D).
Role of Calcineurin in Kv4.2N Mice
The above results suggest that cardiac hypercontractility in early-stage Kv4.2N hearts results from AP prolongation and increased Ca2+ influx through ICa,L. These sustained elevations in [Ca2+]i and ICa,L might also contribute to the transition from hyperfunction to heart failure in the Kv4.2N 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 models19,20⇓ as well as in humans.21 Therefore, calcineurin activity was measured in both control and Kv4.2N mice before (5 to 8 weeks of age) and after (13 to 15 weeks of age) the development of overt heart disease in the Kv4.2N mice. Figure 2, A through C, shows that the calcineurin activity is elevated in Kv4.2N hearts, precedes disease development, and increases progressively (P<0.05) with age. To establish a causal relationship between calcineurin activation and disease initiation, Kv4.2N 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 Kv4.2N mice (HW/BWKv4.2N,Veh=0.57±0.06, n=10; HW/BWKv4.2N,CsA=0.44±0.02, n=8) to control levels. Furthermore, treatment with CsA did not affect body weight in either control or Kv4.2N mice (data not shown).
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 Kv4.2N 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 Kv4.2N hearts are unclear but are consistent with myocyte dropout,6 potentially arising from calcineurin-dependent apoptosis.22 By contrast, CsA treatment of Kv4.2N mice markedly reduced these pathological changes (Figure 4C).
Functional characterization of hearts from 13- to 15-week-old vehicle-treated and CsA-treated Kv4.2N 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 Kv4.2N 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 Kv4.2N 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 Kv4.2N mice reflects a genuine hypercontractility, probably arising from AP prolongation. Although diastolic function was not enhanced in CsA-treated Kv4.2N 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 (τ) (Tables 1 and 2⇓), collectively suggest that normal diastolic function was preserved in CsA-treated Kv4.2N mice.
Effects of Verapamil Treatment on Cardiac Morphology and Ventricular Function
Having established that calcineurin activation is critical for disease progression in Kv4.2N mice, we next tested whether the heart disease process depended on ICa,L. Because the total integrated ICa,L (QCa,L) is increased ≈3-fold as a result of AP prolongation in Kv4.2N myocytes and because the activity of numerous hypertrophic signaling pathways, including the calcineurin pathway, are Ca2+-dependent,25 we examined whether treatment of Kv4.2N 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/BWKv4.2N-Veh=0.51±0.03, n=6; HW/BWKv4.2N-Verap= 0.35±0.02, n=8; HW/BWCON,Veh=0.38±0.01) (Figure 3B) and improved the cardiac morphology of Kv4.2N mice by reducing interstitial fibrosis and cardiomyocyte vacuolization (Figure 4D) compared with vehicle-treated Kv4.2N 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 Kv4.2N 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 Kv4.2N mice sufficiently against disease to preserve the hyperfunctional state.
Expression of SERCA2a and β-MHC and PKC Activation
Reduced expression of SERCA2a and enhanced expression of β-MHC are universal molecular markers of cardiac dysfunction and disease (Figure 5).26,27⇓ In untreated Kv4.2N 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, β-MHC protein expression was elevated 8.2-fold (P<0.05) in untreated 13- to 15-week-old Kv4.2N 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) Kv4.2N mice to levels indistinguishable from control (P>0.05; Figure 5A and 5C).
Because calcineurin activation is associated with a characteristic pattern of PKC isoform activation,14 the membrane translocation patterns of the PKC-α, -ε, and -θ isoforms were examined. The membrane-to-cytosol ratios of the PKC-α and PKC-θ isoforms were increased 6.1- and 2.4-fold, respectively, in Kv4.2N mice relative to controls (Figure 6, A through C). Consistent with a connection between PKC and calcineurin pathways, treatment of Kv4.2N mice with either verapamil or CsA normalized both PKC-α and -θ expression patterns (Figure 6, A through C). In contrast, PKC-ε activation did not differ among any of the groups studied (Figure 6, A and D).
Diminished Ito density and altered AP profile have been associated with heart disease in humans and in numerous animal models.1,2,18,28⇓⇓⇓ This Ito reduction occurs early in the disease process,10 suggesting that Ito may play a role in the initiation of disease.11 Previously, we found that primary Ito reduction achieved by cardiac-restricted overexpression of a truncated Kv4.2N 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 Kv4.2N overexpressing mice.
Consistent with previous studies in rat myocytes,8,9,18⇓⇓ AP prolongation in Kv4.2N transgenic myocytes increased peak [Ca2+]i, supporting the notion that Ito plays an important role in influencing Ca2+ handling and contractility in situations, such as heart disease, in which Ito 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 Ito 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 hypertrophy12,19,20⇓⇓ and during the hypertrophic phase of human heart disease.21 The finding that calcineurin activity was elevated in Kv4.2N mice before the onset of disease prompted us to attempt to prevent disease development by inhibiting ICa,L or calcineurin. The high degree of protection conferred to Kv4.2N transgenic mice by verapamil or CsA suggests that the transition from hyperfunction to failure in Kv4.2N mice is Ca2+-dependent.
Recently, Muth et al13,29⇓ 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 Kv4.2N mice suggests common mechanisms. Indeed, both transgenic models generate chronically elevated ICa,L (by 50% in L-VDCC mice13 and by 200% in Kv4.2N mice) and show activation of Ca2+-dependent PKC-α but not PKC-ε. 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-α and -θ and not -ε, -β, or -λ) 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 Kv4.2N mice.
Regardless of the specific hypertrophic signaling pathways involved, the phenotypes observed in both Kv4.2N 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 Ito downregulation2,10⇓ 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 ICa,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 (ICa,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 ICa,L versus bulk cytosolic Ca2+.
The discrepancy in the phenotypes observed in the Kv4.2N and Kv4.2W362F mice,7 despite similar electrophysiological findings, suggests that secondary effects of Kv4.2N peptide expression,6 in addition to enhanced Ca2+ cycling, may contribute in part to the cardiomyopathy in Kv4.2N mice. Recent studies in our laboratory, however, have demonstrated that overexpression of either Kv4.2N or Kv4.2W362F 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 Kv4.2W362FxKv1.4−/− mice relative to controls,35 suggesting that Ito reduction and AP prolongation do, in fact, contribute to myocyte hypertrophy.
In summary, we have shown that AP prolongation as a result of Ito reduction by overexpression of the Kv4.2N peptide increases contractility in early-stage transgenic myocytes by increasing ICa,L and intracellular [Ca2+]i. Progression to failure then occurs via a Ca2+- and calcineurin-dependent process that is associated with activation of PKC-α and -θ but not -ε and can be inhibited by verapamil and CsA. Although a secondary, indirect effect of Kv4.2N expression may also contribute to disease in these hearts, our data clearly show that ICa,L, [Ca2+]i, and calcineurin are dominant players in the pathology observed after AP prolongation in Kv4.2N 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.
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.
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