Cardiovascular Research

Cardiovascular Research 2003 57(2):320-332; doi:10.1016/S0008-6363(02)00661-2
© 2003 by European Society of Cardiology

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Copyright © 2003, European Society of Cardiology

Overexpression of calcineurin in mouse causes sudden cardiac death associated with decreased density of K⁺ channels

Deli Dong, Yanjun Duan, Jiqing Guo, Dan E Roach, Shauni L Swirp, Li Wang, J.P Lees-Miller, R.S Sheldon, Jeffery D Molkentin and Henry J Duff^*

Cardiovascular Research Group, Department of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1

^* Corresponding author. Tel.: +1-403-220-6841; fax: +1-403-270-0313. hduff{at}ucalgary.ca

Received 11 February 2002; accepted 6 September 2002

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusion References

 
Background: Overexpression of calcineurin in transgenic (TG) mice results in cardiac hypertrophy and unexpected deaths. Methods and results: None of the TG survived beyond 24 weeks (n=38) whereas all of the wildtype (WT, n=47) survived. Prolongation of repolarization preceded the development of sustained pleomorphic ventricular tachycardia and high degree atrioventricular block, which occurred during spontaneous sudden deaths. Since depolarization-activated K⁺ channels contribute dominantly to repolarization in mice, we hypothesized that the TG would decrease these K⁺ currents and that the in vivo administration of cyclosporin A (CsA), a calcineurin inhibitor, would reduce this effect. CsA reversed cardiac hypertrophy: capacitance measurements of WT left ventricular myocytes (127±7 pF; n=45) and CsA-treated TG (129±14 pF; n=17) were significantly lower than in placebo-treated TG (220±11 pF; n=41; P<0.001 by ANOVA). Independent of whether the data fit a bi- or a tri-exponential model, the density of I_tof was significantly reduced in TG versus WT and CsA reversed this effect. While I_tos and I_Kslow were also reduced in TG, CsA does not reverse this change because long-term in vivo CsA treatment of WT also reduces I_tos and I_Kslow. To assess whether the decreased ‘repolarization reserve’ contributed to arrhythmogenesis, the residual I_Kr was blocked by dofetilide precipitating pleomorphic ventricular tachycardias. Conclusion: Since the downregulation of I_tof was observed with overexpression of calcineurin and was also reversed by the calcineurin inhibitor CsA, we conclude that downregulation of I_tof is a consequence of calcineurin overexpression.

KEYWORDS Tau₁, relates to the I_Kslow component; Tau₂, relates to the I_tos component; Tau₃, relates to the I_tof component; TG, transgene; TGCsA, transgene treated with CsA; WT, wildtype; WTCsA, wildtype treated with CsA

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This article is referred to in the Editorial by R. Wolk (pages 289–293) in this issue.

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusion References

 
Calcineurin is a calcium-activated phosphatase that dephosphorylates a cytosolic protein, NF-AT3, which translocates into the nucleus to interact with the transcription factor GATA-4 thus activating a range of genes involved in cardiac hypertrophy [1]. Molkentin et al. overexpressed a constitutively active calcineurin in transgenic mouse (TG) hearts and observed massive hypertrophy followed by fibrosis, congestive heart failure and unexpected deaths [1]. One of the objectives of this study was to define the mechanism of these unexpected deaths.

Most [2–20] but not all studies [8–12] have reported that the calcineurin inhibitor, CsA, inhibits the development of pressure-overload-induced and catacholamine-induced hypertrophy. Pressure-overload cardiac hypertrophy also increases calcineurin protein content and enzyme activity [20]. Pressure-overload cardiac hypertrophy results in downregulation of ion channels including potassium channels and the sarcoplasmic reticulum calcium cycling proteins [21–24]. Yatani et al. have previously examined the effects of overexpression of calcineurin on the I_{Ca-L and T types} in ventricular myocytes [25]. Accordingly, the present study focuses on changes in the density and function of potassium channels in the calcineurin-overexpressing TG versus wildtype mouse (WT). Transgenic overexpression of any protein could produce non-specific cellular toxicity. To assess whether downregulation of K⁺ channels was a consequence of calcineurin overexpression, the K⁺ current densities and function were evaluated after long-term in vivo treatment with the calcineurin inhibitor, CsA (12 mg/kg, subcutaneously).

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusion References

 
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

ICR (WT) and ICR (TG) female mice were studied at 31–33 days of age, since hypertrophy was well established by this time [1]. Southern blots were used to separate WT and TG [1]. Electrophysiologic studies were performed on only one mouse line since Molkentin et al. have reported that all lines have identical cardiac phenotypes [1].

2.1 Recording electrocardiograms
ECGs were recorded in conscious, unsedated mice [26] using a restraining tube with perforations that allow the limbs to fall through. ECG electrodes (silver-silver chloride) were applied to the skin. The QT interval was measured as the time when repolarization returned to the isoelectric point. The isoelectric point [26,27] was defined as the position just before the P-wave [27]. Similarly, Mitchell et al. [28] assigned the end of the T-wave as the isoelectric point, which follows a terminal negative T-wave vector in the surface ECGs. The rate correction has been previously reported [26,28].

2.2 Long-term in vivo treatment with CsA
Dose and time–response relationships to a range of CsA concentrations up to 25 mg/kg per day [1] were performed. Treating 7-day-old mice at this dose caused progressive weight loss and eventually death. The final strategy consisted of randomly assigning 7-day-old TG to placebo or CsA [1] at 12 mg/kg per day, the maximum well tolerated dose.

2.3 Whole-cell patch clamp recording
Myocytes were prepared and macroscopic K⁺ currents were recorded at room temperature using previously reported methods [29]. All myocytes were obtained from the apex of the left ventricle. CdCl₂ (0.3 mmol/l) blocked the L-type Ca²⁺ current. The Axoclamp 200 amplifier was modified by Axon Instruments to allow capacitance compensation of a wider range of cells with capacitances between 1 and 1000 pF. Increased capacitance in hypertrophied myocytes raises the possibility of potential spatial-clamp errors. The determinants of spatial-clamp errors are the magnitude and rapidity of the kinetics of the ion current and series resistance compensation [30,31]. Large currents with extremely fast kinetics such as the sodium current are sensitive to spatial-clamp errors. However, the depolarization activated K⁺ currents in this study were smaller and slower than the sodium channel. Furthermore, the currents in transgenic myocytes are smaller than WT. In addition, Bouchard et al. [31] have emphasized the importance of series resistance in reducing spatial-clamp errors. Accordingly, series resistance was controlled to no more than 5 M and was compensated to at least 90% in this study. If this degree of compensation could not be achieved, the cells were discarded. Series resistance was regularly checked to ensure there were no variations with time. In the case where series resistance increased during the recording, the experiment was discarded [29]. Cell capacitance and series resistance were calculated from uncompensated capacity current transients elicited by a 10-mV hyperpolarizing voltage step from a holding potential of –80 mV.

The sodium current did not confound our measurements of I_to. The reversal potential of the sodium current was approximately +50 mV under our experimental conditions. At the reversal potential, we observed the same decrease in density of I_to as seen at 0 to +40 mV. At positive potentials, the sodium current activation time is accelerated with activation times substantially less than 1 ms, whereas I_to activates in 2–5 ms. In order to provide experimental evidence, we have recorded the density of I_to before and after administration of TTX at 30 µM, a concentration that virtually abolished the cardiac sodium current. We observed no substantial change (<5% reduction) in density of I_to (Fig. 1; n=3 cells from one mouse).

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 The TTX-sensitive sodium current does not confound measurements of Ito. (Panel A) Representative examples of the peak Ito recorded before and after TTX 30 µM/l. (Panel B) TTX had no significant effect on the Ito current densities (n=3).

 
Current clamp recordings were used to record action potentials in single cardiac myocytes.

2.4 Data analysis
A rigorous and unbiased method to establish the optimal fit to a multiexponential function using the Pade-Laplace transformation has been reported [32,33]. This method was utilized to determine the appropriate number of exponential fits to the inactivation records using custom software created in MATLAB [32,33]. Essentially, this technique numerically estimates the Taylor series coefficients for the Laplace transform of the current decay data. These coefficients are then used to construct successively higher-order Pade approximants of the Laplace transform. Laplace transforms of exponential series can be completely described by the location and magnitude of their complex poles. Thus, for successively higher-ordered Pade approximants (up to order 6), the number of exponentials present in the decay data equals the maximum number of real poles with non-trivial magnitudes.

Table 1 compares Chebyshev fits of TG data to tri- and bi-exponential fits when the Pade-Laplace transformation had indicated a bi-exponential process. Gross increases (50–100-fold) in the mean voltage-dependent scatter (mean standard error of the estimates) of A₁, tau₁, A₂, and tau₂ and nonsense terms are observed when inappropriately applying the tri-exponential fits. These data indicate that gross errors (in bold) occur when the results of Pade-Laplace are ignored. Another type of error can occur if the results of the Pade-Laplace are disregarded. For example, Fig. 2 shows superimposed fits of WT data to bi- (panel A) and a tri-exponential (panel B) processes when Pade-Laplace indicated a tri-exponential process. Inappropriate bi-exponential fits missed the fast component (I_tof). Based on these data, the results of the Pade-Laplace transformation appear to be reliable.

View this table: [in this window] [in a new window]   Table 1 Representative mean data of fitting of inactivation for TG

 

View larger version (7K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Errors are made when a bi-exponential fit is applied to an exponential decay established to be tri-exponential by the Pade-Laplace transformation. Superimposed fits of WT data to bi- (panel A) and tri-exponential (panel B) fits when Pade-Laplace indicated a tri-exponential process are shown. Inappropriate bi-exponential fits missed the fast component (arrow). The results of the Pade-Laplace transformation appear to be reliable.

 
The signal average of the mouse ECG was determined by aligning all cardiac cycles to a common point in the ECG waveform. To do this, we created software in MATLAB designed to perform a wavelet transform of the ECG using the second derivative of the Gauss function as our wavelet (Gauss support [–5,5] and wavelet scale 25 ms). Each R-wave produced a large magnitude local minimum in the wavelet transform space that could be easily localized using a threshold. The ECG for each cardiac cycle was defined as those voltages within a sampling window starting 35 ms prior to the first R-wave and ending 35 ms prior to the next R-wave. Through this process, the means of the compiled cycles for each 60-s recording were calculated.

Data are presented as mean±S.E.M. One-way ANOVA with Dunnett's multiple range test was used with a P<0.05 considered significant. Data analysis was performed in two ways. In one analysis, the data from each cell were considered as an independent sample. In this case, the n value indicates the number of cells analyzed. In the alternative analysis, the mean of all the data for a single animal, at a single potential was taken, and only this value considered for each animal. As opposed to the previous analysis, n value here is related to the number of animals. For example, if seven cells were examined from one animal, only the single mean value for that individual animal was included in the statistical analysis.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusion References

 
3.1 Survival of transgenic mice
The survival rate of WT (open symbol) versus TG (closed symbol) is shown in Fig. 3A. None of 47 WT died, whereas none of 38 TG survived beyond 24 weeks. Deaths in TG were unexpected. By serendipity (or experience), two free-roaming mice were observed to suddenly become still, in a huddled position, and ignored their littermates but were not unconscious since they responded to touch with movement. ECGs were immediately obtained for these mice and recorded spontaneous deaths at 10 min and 45 min after the onset of recording. The ECGs obtained during these spontaneous sudden deaths are shown in panels C and D of Fig. 3. In one animal, age 8 weeks (panel C), there were recurrent episodes of sustained pleomorphic ventricular tachycardia that eventually self-terminated and were replaced by high degree AV block and ultimately asystole. In the other animal, age 11 weeks (panel D), the first observed ECG abnormality was high degree AV block with a mean ventricular response of 943±509 ms. This was followed by slow non-sustained VT with a cycle length of 174±66 ms and eventually slow agonal rhythm and asystole.

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Survival of transgenic mice (, n=38) is compared to that of wildtype mice (, n=47). Panel A shows survival of TG versus WT. None of the 47 WT died, whereas none of the TG survived beyond 24 weeks (n=38). Panel B shows the time-dependent increase in the proportion of animals manifesting non-sustained ventricular tachycardia and Wenkebach block. Panels C and D show episodes of spontaneous sudden deaths. In panel C, recurrent episodes of pleomorphic VT self-terminated were replaced by high degree AV block and asystole. In the other animal (panel D) episodes of high degree AV block were replaced by slow ventricular tachycardia (mean cycle length 174±66 ms) and ultimately asystole.

 
3.2 Calcineurin overexpression prolongs repolarization in vitro and QT intervals in vivo
Fig. 4 shows representative examples and mean action potential durations in WT and TG myocytes. Repolarization was significantly prolonged in TG (P<0.05). Fig. 5, panel A, shows representative examples of signal averaged ECGs recorded from WT and TG illustrating the change in the configuration of the ECG and the prolongation of the RR and QT intervals observed in the TG. The mean intervals are shown in panel B. The PR interval was also prolonged but Wenkebach block was frequently present so the PR interval was not constant.

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Action potential phenotype of TG versus WT. Representative examples of action potentials recorded in single cardiac myocytes from WT and TG are shown in panel A. Mean action potential durations recorded at 50 and 90% repolarization are shown in panel B. *P<0.05 by unpaired Student's t-test.

 

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 ECG phenotype of TG versus WT. Representative examples of signal averaged ECGs recorded in WT and TG are shown in panel A. The isoelectric position is labelled and shown as the fine horizontal line. The boxed sections of the ECG in the left panels are magnified in the right panels. The arrows show the end of the T-wave. Mean ECG interval data are shown in panel B.

 
3.3 Measures of cardiac hypertrophy
Mice were euthanized at 31–33 days. The mean ratio of wet heart weight/body weight of TG (0.02±0.00) was significantly greater than WT (0.007±0.001; P<0.01). In TG treated from day 7 with CsA, this ratio was 0.01±0.004, a value significantly less than for placebo-treated TG. Capacitance of WT myocytes (127±7 pF; n=45 cells from five mice) was significantly less than for TG (220±11 pF; n=41 cells from six mice; P<0.001). The capacitance of CsA-treated TG was 129±14 (n=17 cells from four mice), a value not different from WT.

3.4 Components of peak outward currents
For WT, the Pade-Laplace transformation indicates that the best fit is achieved with a tri-exponential fit in 80% of the 270 traces in keeping with previous studies [34,42]. In WT treated with CsA, 71% of the 210 traces were fit to a tri-exponential equation. In contrast for TG, a bi-exponential fit was best for 70% of the 246 traces. In TG treated with CsA, 77% of 102 traces were best fit to a bi-exponential model.

For WT, 45 cells from five mice with a median six cells per mouse; for TG, 41 cells from six mice with a median of five cells per mouse; for CsA-treated TG, 17 cells from four mice with a median of five cells per mouse; and for CsA-treated WT 49 cells from four mice with a median of 13 cells per mouse were assessed. All the mice evaluated were females.

The nomenclature of I_tof, I_tos, and I_Kslow stems from earlier work evaluating genetically modified mice containing knockouts of Kv4.X, Kv1.4, Kv2.1, Kv1.5 gene groups [35–44]. The results showed that each gene contributed to a different component of the transient outward current. Namely, Kv4.X contributed to I_tof, Kv1.4 to I_tos, Kv2.1 and Kv1.5 to I_Kslow. We have adopted this nomenclature for the individual components in this study.

Table 2 shows the mean time-constants of the components of the depolarization-activated K⁺ currents. In TG cells best fit to the bi-exponential equation, CsA significantly slowed the slow tau₁ component (I_Kslow), but had no significant effect on WT. In addition, CsA significantly accelerated the fast tau₂ component (I_tof) but only in TG. In cells that are best fit to a tri-exponential equation, the tau₂ component was modestly altered but not at all voltages. In summary, CsA modestly but significantly alters the fast and slow taus in TG.

View this table: [in this window] [in a new window]   Table 2 Inactivating components of Ito

 
3.5 Densities of the components of the depolarization-activated K⁺ currents
For the data that were best fit to a tri-exponential model, the mean current densities for I_tof, I_tos and I_Kslow in TG versus WT myocytes are shown in Fig. 6, panels A, B and C, respectively. TG manifested a significant decrease in the densities of all three components. CsA treatment of TG restored the density of I_tof to values similar to or greater than WT. Similar results were observed in the data best fit to a bi-exponential model as shown in panels D and E. In summary, the density of I_tof is restored by CsA treatment whereas the densities of the other components are not.

View larger version (31K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 TG decreases densities of all components of Ito but only Itof is restored by CsA. Panels A, B and C show the density of Itof, Itos and IKslow, respectively, in cells in which the Pade-Laplace transformation indicated a best fit to a tri-exponential model with WT (open symbols) and TG (closed symbols; *P<0.01) and TG treated with CsA (closed symbols with a ‘+’). Panels D and E show the density of Itof and IKslow, respectively, in cells in which the Pade-Laplace transformation indicated a best fit to a bi-exponential model. *P<0.05; NS, non-significant.

 
For data best fit to a tri-exponential model, the density of I_tof in CsA-treated WT was similar to that in placebo-treated WT (Fig. 7, panel A), but the densities of I_tos and I_Kslow were significantly reduced in CsA-treated WT (panels B and C). Similar results were observed in the data best fit to a bi-exponential model (panels D and E).

View larger version (31K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Effects of long-term in vivo CsA on WT. Mean current densities for Itof (panel A), Itos (panel B) and Islow (panel C) are shown for data best fit to a tri-exponential model while panels D and E show data for data best fit to a bi-exponential equation. Placebo-treated WT data are shown in open symbols and CsA-treated WT data are shown with a .

 
Due to the fact that an unequal number of cells were sampled from each mouse and to avoid the possibility that data from a single mouse could dominate the statistical analysis, the ANOVA analysis was repeated when only a single mean data point (at each voltage) was included from each mouse. Using this analysis, overexpression of calcineurin (CN) significantly decreased the densities of I_tof, I_tos and I_Kslow compared to WT. For example, at +40 mV I_tof was 30±6 pA/pF in WT (n=5 mice) and 15±2 pA/pF in TG (n=6 mice), 47±11 pA/pF in TG treated with CsA (n=4 mice) and 30±5 pA/pF (n=4 mice) in WT treated with CsA (P=0.002 by ANOVA). Multiple range testing indicates that I_tof is significantly reduced in TG compared to WT and is significantly increased by treatment of TG with CsA. Similarly at +40 mV, density of I_Kslow was 18±3 pA/pF in WT (n=5 mice), 9±1 pA/pF in TG (n=6 mice), 8±1 in TG treated with CsA (n=4) and 14±2 pA/pF (n=6) in WT treated with CsA (P=0.002 by ANOVA). Multirange testing indicates that I_Kslow was significantly reduced in TG but was not increased by treatment with CsA. However, using this analysis, the reduction of I_Kslow in WT treated with CsA was not significantly different than WT. For I_tos, different results were observed using this statistical analysis. At +40 mV, I_tos was 14±4 pA/pf in WT (n=5 mice) and 8±2 pA/pF in TG (n=6 mice), 4±1 pA/pF in TG treated with CsA (n=4 mice) and 9±1 pA/pF (n=4 mice) in WT treated with CsA. The only significant difference by multirange testing was between WT and TG. With the exception of I_tos, the statistical results are similar, independent of whether each cell is considered an independent sample or whether only one single mean sample was included from each mouse.

3.6 Steady-state inactivation
Fig. 8, left panels shows representative current traces elicited by the double-pulse protocol for WT (panel A) and TG (panel B). When a pre-pulse progressively depolarized the cell membrane from –100 mV to +20 mV, the amplitude of the peak currents progressively decreased suggesting decreased channel availability with depolarization. The magnitude of each component was normalized to the value of the current after the prepulse to –100 mV. Normalized data (open symbols for WT and closed symbols for TG) for individual components were fit to Boltzmann equations shown in the right panels. The data are best fit to a single Boltzmann for I_tof. Mean values of half inactivation potential (V_1/2) and slope factors (k) for WT were –36 mV, k = –8; n=20 and similar values for TG were –36 mV, k = –5, n=20; and for TG treated with CsA, values were –36 mV and k = –4, n=14. Boltzmann fits of normalized data for I_Kslow showed mean V_1/2 and slope factors (k) for WT of V_1/2=–33 mV, k₁=–9, n=20 and for TG of V_1/2=–36 mV, k₁=–5, n=20. The V_1/2 and slope factors for all of the inactivation components were not altered by CsA treatment.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 Steady-state inactivation in TG and WT myocytes. The left panels show representative examples of the currents evoked by the protocol shown in the inset. The right panels show normalized data for each individual inactivating component fitted to Boltzmann equations with WT (open symbols) and TG (closed symbols) Itof circles, Itos triangles and IKslow in squares.

 
3.7 Recovery from inactivation
Fig. 9 shows representative examples of such recordings for WT (panel A) and TG (panel B). The time-course of recovery of I_tof, I_tos and I_Kslow components are shown in the right panels. The time-course of recovery from inactivation for I_tof (right panels) were best fit by bi-exponential equations in WT and TG with fast time constants of 36 and 49 ms, respectively, and slow time constants of 910 and 1250 ms, respectively (Fig. 8). Similarly, the time-courses of recovery from inactivation of I_Kslow (right panels) were best fit by a bi-exponential equations in both WT and TG with fast time constants of 292 and 185 ms, respectively (P<0.05) and slow time constants of 2522 and 1595 ms, respectively (Fig. 9). CsA treatment had no significant effect on the kinetics of recovery from inactivation of I_tof, I_tos, or I_Kslow.

View larger version (25K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 9 Time-course of recovery from inactivation. A double-pulse protocol was used to measure recovery from inactivation. The first pulse (P1) depolarized the membrane from –80 to +50 mV followed by a second pulse (P2) from –80 to +50 mV at variable interpulse intervals to determine the fraction of current recovered at a given interpulse interval. The left panels show representative examples of the currents evoked by the protocol shown in the inset. The amplitude of the individual inactivating components elicited by P2 was normalized to that of the P1 pulse and plotted against the interpulse intervals (right panels). Itof is shown as for WT, for TG; Itos is shown as for WT and IKslow is shown as for WT, and for TG.

 
3.8 Repolarization reserve and arrhythmogenesis
Components of I_to are the dominant repolarizing currents in mice but their function is downregulated in TG so residual repolarizing currents might take on important physiologic roles. We reasoned that the dofetilide-sensitive I_Kr contributes to a safety factor for repolarization. Fig. 10 shows representative examples of arrhythmogenic responses to dofetilide (0.5 mg/kg, i.p.) in 29–32-day-old TG. None of the animals developed sudden cardiac death after dofetilide treatment (Fig. 10). These data indicate that the residual dofetilide-sensitive currents are physiologically important for repolarization in TG and pharmacologic blockade of these currents can precipitate serious arrhythmias.

View larger version (51K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 10 Dofetilide-induces pleomorphic ventricular tachycardias in TG. Of the eight TG studied, dofetilide induced spontaneous pleomorphic ventricular tachycardia in three (panel A) whereas ventricular tachycardia was induced in not one of eight WT mice. Two other TG had non-sustained ventricular tachycardia both before and after dofetilide (panel B) and three mice (panel C) did not show arrhythmias either before or after dofetilide.

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusion References

 
The new information provided by this study includes the following facts: (i) overexpression of calcineurin results in sudden cardiac arrhythmic deaths that are preceded by development of high degree AV block (bradycardia) associated with pleomorphic ventricular tachycardia and prolonged QT intervals; (ii) the density of I_tof, I_tos and I_Kslow is significantly reduced by overexpressing calcineurin in heart; (iii) CsA, the calcineurin inhibitor, reversed the effects of overexpressing calcineurin on I_tof but not on I_tos or I_Kslow; and (iv) in WT, CsA decreased the density of I_Kslow and I_tos with no effect on density of I_tof. Since CsA has electrophysiologic effects even in WT, its effects on the TG reflect a superimposition of reversal of hypertrophy by CsA and the direct effects of CsA on these potassium currents.

The clinical relevance of this work is that when components of I_to are significantly reduced by disease, the repolarization reserve is decreased and any residual current may play a more important physiologic role in a diseased ventricle. Dofetilide block of I_Kr in TG provokes life threatening ventricular arrhythmia providing an important link between potassium channel downregulation and propensity for sudden death.

4.1 Sudden death in TG is associated with high degree AV block/pleomorphic VT
Unexpected death in TG has been previously reported [1]. We provide evidence that these are sudden cardiac deaths. We propose that a number of electrophysiological features must occur in unison to allow the development of sudden cardiac death. The acquired long QT syndrome observed in this TG, produced at least in part by downregulation of components of I_to, is a part of the substrate predisposing to sudden death. However, this feature is not sufficient in and of itself to cause arrhythmogenesis. Bradycardia-induced exaggeration of the prolonged QT interval appears to contribute to the trigger for the ventricular arrhythmias. These electrophysiologic features suggest conditions for early after depolarization. The observation that dofetilide can precipitate arrhythmogenesis indicates that triggers that block the residual repolarizing currents further deplete repolarization reserve and predispose to early after depolarizations. The other electrophysiologic feature that can predispose to early after depolarizations is an increase in the L-type calcium currents. Yatani et al. previously reported this electrophysiologic feature in this TG [25]. Consequently, none of these electrophysiologic features taken in isolation may be sufficient to cause arrhythmias.

We propose that downregulation of I_to does contribute to the milieu predisposing to sudden cardiac deaths. However, downregulation of I_to in isolation may not be sufficient for arrhythmogenesis. We speculate that arrhythmogenesis results from a complex superimposition of downregulation of potassium channels, intracellular calcium-overload, activation of the Na⁺/Ca²⁺ exchanger, bradycardia, conduction defects, fibrosis and the cell-to-cell uncoupling associated with structural heart disease. These features taken in synchrony would be required to produce sudden cardiac death. The observation that dofetilide has no effect on WT but precipitates serious ventricular arrhythmias in the TG is in keeping with the idea that a decrease in the repolarization reserve is also a contributor to complex substrate for arrhythmogenesis. However, our data does not exclude the contribution, even a dominant contribution, by other mechanism(s), they simply provide evidence for a contribution of decreased repolarizing currents to the propensity for arrhythmias.

4.2 Comparison to previous studies evaluating immunosuppressants
Previous studies have reported the in vitro effect of FK506 on K⁺ currents in mouse ventricle [45]. The inactivating components of I_to were evaluated in that study using 300- and 1000-ms pulses in contrast to the 4-s pulse used in the present study. The I_tos component was not observed in that study presumably because of the short pulse interval. Moreover, the tau of I_Kslow (tau=1700 ms) recorded in the present study is longer than the entire pulse duration studied by Du Bell et al. [45]. Their measurement of the sustained (pedestal) component likely includes the slowly decaying I_Kslow. In contrast to the study of FK-506, we find that long-term in vivo treatment with CsA does not decrease I_tof.

4.3 Limitations
Overexpression of calcineurin downregulates potassium channels but this is associated with cardiac hypertrophy and fibrosis. The present study does not discriminate between whether the downregulation of the potassium channels is directly related to calcineurin or is secondary to the hypertrophy and fibrosis. However, the fact that downregulation of potassium channels is common to many forms of cardiac hypertrophy suggests that disease, as a consequence of calcineurin overexpression, is the cause of this downregulation.

Many of the sudden cardiac deaths occurred in mice older than those studied in the patch clamp studies. Consequently, the electrophysiologic data obtained at 31–33 days may not be extrapolated to the electrophysiologic substrate associated with the sudden deaths. It has not been possible to obtain high quality myocytes from calcineurin mice older than those studied herein. Therefore our data must be interpreted with caution. Female mice were studied. The results cannot be extrapolated to male mice.

	5. Conclusion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusion References

 
Overexpression of calcineurin decreases the density and function of the depolarization-activated K⁺ currents and density of I_tof is restored by CsA treatment.

Time for primary review 22 days.

	Acknowledgements

 
This study was funded by the Andrew Family Professorship in Cardiovascular Science and the Canadian Institute of Health Research.

	References

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusion References

 

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