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(Circulation. 2001;104:1657.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Electrical Remodeling in Pressure-Overload Cardiac Hypertrophy

Role of Calcineurin

Zhengyi Wang, MD; William Kutschke, MS; Kenneth E. Richardson, BS; Mohsen Karimi, MD; Joseph A. Hill, MD, PhD

Departments of Internal Medicine (Z.W., W.K., J.A.H.), Pharmacology (J.A.H.), and Surgery (M.K.), and the Interdisciplinary Graduate Program in Molecular Biology (K.E.R., J.A.H.), University of Iowa College of Medicine, and the Department of Veterans Affairs (J.A.H.), Iowa City, Iowa.

Correspondence to Joseph A. Hill, MD, PhD, Cardiovascular Division, University of Iowa College of Medicine, E318GH, UIHC, 200 Hawkins Dr, Iowa City, IA 52242-1081. E-mail joseph-hill{at}uiowa.edu

	Abstract

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Background— Myocyte hypertrophy accompanies many forms of heart disease, but its contribution to electrical remodeling is unknown.

Methods and Results— We studied mouse hearts subjected to pressure overload by surgical thoracic aortic banding. In unbanded control hearts, action potential duration (APD) was significantly longer in subendocardial myocytes compared with subepicardial myocytes. Hypertrophy-associated APD prolongation was significantly greater in subendocardial myocytes compared with subepicardial myocytes, indicating stress-induced amplification of repolarization dispersion. To investigate the underlying basis, we performed voltage-clamp recordings on dissociated myocytes. Under control unoperated conditions, subendocardial myocytes exhibited significantly less transient outward current (I_to) than did subepicardial cells. Hypertrophy was not associated with significant changes in I_to, sustained current, or inward rectifier current densities, but peak L-type Ca²⁺ current density (I_Ca,L) increased 26% (P<0.05). Recovery from I_Ca,L inactivation was accelerated in hypertrophied myocytes. Inhibition of calcineurin with cyclosporin A prevented increases in heart mass and myocyte size but was associated with an intermediate APD. The hypertrophy-associated increase in I_Ca,L and the accelerated recovery from inactivation were blocked by cyclosporin A.

Conclusions— These data reveal regional variation in the electrophysiological response within the left ventricle by way of a mechanism involving upregulated Ca²⁺ current and calcineurin. Furthermore, these results reveal partial uncoupling of electrophysiological and structural remodeling in hypertrophy.

Key Words: calcium • electrophysiology • action potentials • arrhythmia • hypertrophy

	Introduction

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Faced with stressful stimuli, cardiac myocytes mount a growth response that culminates in hypertrophy. Cardiac hypertrophy is associated with a significantly increased risk of cardiovascular morbidity and mortality, much of which stems from electrical remodeling and arrhythmogenesis.^1,2 In the left ventricle (LV), electrical remodeling is usually manifested as action potential prolongation, which predisposes to early and late afterdepolarizations³ and underlies the propensity to arrhythmia, syncope, and sudden death.

Considerable progress has been reported recently in elucidating the molecular pathogenesis of hypertrophy (as reviewed recently in Olson and Williams⁴, and Molkentin⁵), but our understanding of the mechanisms of electrical remodeling in hypertrophy is limited. Development of new antiarrhythmic therapies will require identification of disease-specific mechanistic pathways leading to action potential prolongation.

In humans, electrophysiological heterogeneity within the ventricular wall underlies a number of physiological (upright T wave on the surface ECG) and pathological (torsades de pointes, J wave of Osborne, U waves) processes.^6,7 In many species, this dispersion of repolarization stems from the relatively brief action potential duration (APD) of subepicardial myocytes, which manifest a high-density transient-outward current (I_to).^7–9 Although genetic models in mice have been instrumental in dissecting the electrical anatomy of the ventricular action potential,^10–12 it is not known whether transmural electrical heterogeneity exists in the mouse. Furthermore, it is not known whether hypertrophy alone, a process associated with a markedly increased risk of sudden death, is associated with heightened spatial heterogeneity of electrical remodeling.

In this study, we investigated the electrophysiological phenotype of myocardial hypertrophy in a model with no LV dysfunction.¹³ We addressed 3 questions: (1) Is hypertrophy associated with electrical remodeling, and if so, what is the underlying electrophysiological basis? (2) Does hypertrophic transformation amplify the regional variation of recovery of electrical excitability in the LV? (3) What is the role of a recently described calcineurin transcriptional pathway?

	Methods

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Pressure-Overload Hypertrophy Model
Male mice (C57BL6, 6 to 8 weeks old) were subjected to pressure overload by thoracic aortic banding (TAB).¹³ Some mice were subjected to a sham operation in which the aortic arch was visualized but not banded. Perioperative (24-hour) mortality was <10%. On the morning of postoperative day 1, TAB or sham-operated mice were randomized to receive either cyclosporin A (CsA) 25 mg/kg or vehicle (Veh) subcutaneously twice daily.

Myocyte Isolation
LV cardiomyocytes were isolated by using a protocol slightly modified from that of Xu et al.¹⁴ In brief, after retrograde perfusion with Ca²⁺-free buffer followed by collagenase solution, the apex was removed and the LV midwall was divided into subendocardial- and subepicardial-enriched fractions. After trituration, cells were plated and studied within 6 to 8 hours.

Electrophysiological Recordings
Myocytes were superfused at 1 to 2 mL/min (21°C to 23°C) with Tyrode’s solution containing (in mmol/L) 137 NaCl, 5.4 KCl, 1.0 MgCl₂, 1.0 CaCl₂, 10.0 dextrose, and 10.0 HEPES (pH 7.4). Calcium-tolerant quiescent myocytes with a typical rod-shaped appearance and clear cross striations were chosen for experimentation. Borosilicate glass capillaries (7052 glass, 1.65-mm OD, A-M Systems) were prepared with tip resistances of 1.5 to 3.0 M when filled with a solution containing (in mmol/L) 137.0 KCl, 1.0 MgCl₂, 0.5 CaCl₂, 10.0 HEPES, 5.0 EGTA, 5.0 Mg₂-ATP, 5.0 sodium creatine phosphate, and 0.5 GTP-Tris (pH 7.2). Tip potentials were compensated before the pipette touched the cell. After whole-cell voltage clamp with a gigaohm seal was obtained, whole-cell membrane capacitance was calculated as the time integral of the capacitative response to a 10-mV hyperpolarizing step. Cells with significant leak current (100 pA) were rejected (20%). When whole-cell currents (depolarized every 15 seconds) were measured, series resistance and membrane capacitance were compensated electronically 85%.

To record depolarization-activated K⁺ currents, the L-type Ca²⁺ current (I_Ca,L) was inhibited with 2 mmol/L CoCl₂. This agent was chosen because other blockers have nonspecific actions on the Na⁺ current (Cd²⁺) or I_to (dihydropyridines). A holding potential of -50 mV was used to inactivate rapid Na⁺ currents.¹⁵ To record I_to and I_sus, 350-ms depolarizing pulses (-40 to +60 mV) from a holding potential of -50 mV were applied. I_sus was defined as the current remaining at the end of the pulse. I_to was defined as the difference between peak current (I_peak) and I_sus. To measure I_Ca,L, cells were continuously superfused with a solution containing (in mmol/L)¹⁶ 2.0 CaCl₂, 1.0 MgCl₂, 135.0 tetraethylammonium chloride, 5.0 4-aminopyridine, 10.0 glucose, and 10.0 HEPES (pH 7.3). The pipette-filling solution contained (in mmol/L) 100.0 cesium aspartate, 20.0 CsCl, 1.0 MgCl₂, 2.0 Mg₂-ATP, 0.5 GTP, 5.0 EGTA, and 5.0 HEPES (pH 7.3).

Statistical Methods
Averaged data are reported as mean±SEM. Sample sizes are listed as n=x/y to denote x cells from y mice. Statistical significance was analyzed with a Student’s unpaired t test or 1-way ANOVA followed by Bonferroni’s method for post hoc pairwise multiple comparisons. Quality of exponential fits was analyzed with a ² test statistic.

	Results

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Recent studies have revealed heterogeneity in the electrophysiological architecture of the mammalian heart in physiological and pathological conditions.⁶ In large mammals, the transient outward current (I_to) is more prominent in subepicardial myocytes than in other cells.^7,8 To investigate this in mice, short-axis slices of LV were microdissected into subepicardial- and subendocardial-enriched fractions. Current-clamp action potentials recorded in subepicardial (Figure 1A) and subendocardial (Figure 1C) myocytes from unoperated control mice were similar in morphology, displaying relatively little phase 2 plateau. APD in subendocardial myocytes was statistically significantly greater than that recorded in subepicardial cells (Table 1).

View larger version (18K): [in this window] [in a new window]   Figure 1. Representative action potentials recorded from ventricular myocytes isolated from subepicardial (A) or subendocardial (C) fraction of control unoperated LV. Outward K+ currents recorded by stepping from -50 mV to voltages between -40 to +60 mV in myocytes isolated from subepicardial (B) or subendocardial (D) fraction. Action potentials recorded from myocytes isolated from sham-operated mice were identical.

View this table: [in this window] [in a new window]   Table 1. APDs and Whole-Cell Current Densities Measured at +40 mV

To investigate the basis for the differences in APD, whole-cell depolarization-activated K⁺ currents were recorded under conditions in which inward Na⁺ and Ca²⁺ currents were inhibited (Table 1). In the majority of myocytes (16/22 cells from 10 mice, 73%) isolated from the subepicardial fraction of unoperated control LV, voltage-clamp recordings revealed a large I_to (Figure 1B). In contrast, the majority of cells (15/23 cells from 11 mice, 65%) isolated from the subendocardial fraction did not reveal substantial I_to (Figure 1D). In contrast, I_sus densities were similar in these 2 cell fractions.

Role of Calcineurin in Spatial Dispersion of Hypertrophic Electrical Remodeling
To determine whether hypertrophic electrical remodeling is heterogeneous within the mouse LV, mice were subjected to surgical aortic banding for 3 weeks. Heart weight normalized to body weight increased 59% (P<0.05) in TAB+Veh-treated hearts compared with controls (Sham+Veh; Table 2). Isolated ventricular myocytes were enlarged significantly in TAB+Veh hearts compared with Sham+Veh hearts assayed in terms of whole-cell membrane capacitance (increase of 33%, P<0.01) and 2-dimensional cell surface area (increase of 52%, P<0.01; Table 2). Within each treatment group, cell capacitances and 2-dimensional cell surface areas were similar in subendocardial and subepicardial myocytes (Table 2).

View this table: [in this window] [in a new window]   Table 2. Heart Mass, Myocyte Surface Area, and Whole-Cell Capacitance for Each Treatment Group

We have shown previously that calcineurin inhibition with CsA blocks the increases in heart mass and myocyte size associated with pressure overload in this model.¹³ Because it is not known whether the electrophysiological and structural phenotypes of hypertrophy are causally linked, we examined whether they could be dissociated by calcineurin inhibition. To control for the influence of hypertrophic growth on electrophysiological remodeling, mice were randomized on the morning after TAB or sham operation to receive either CsA (25 mg/kg SC BID) or vehicle. In hearts exposed to CsA (TAB+CsA or Sham+CsA), heart mass, myocyte surface area, and myocyte capacitance were not significantly different from baseline (Table 2), thereby confirming the efficacy of hypertrophy abrogation. Calcineurin phosphatase activity in the sham-operated LV was 349±18 pmol · mg^-1 · min^-1 (n=8), which was not statistically significantly different from TAB (260±40 pmol · mg^-1 · min^-1, n=7, P=NS). In the presence of CsA, however, calcineurin activity (49±7 pmol · mg^-1 · min^-1, n=7) was significantly diminished compared with sham-operated (P<0.01) or TAB (P<0.01) conditions.

Current-clamp action potentials revealed significant differences in the electrophysiological response to pressure overload in cells derived from 2 regions of the LV wall (Table 1). Action potentials recorded in hypertrophied (TAB+Veh) subepicardial myocytes (Figure 2A) were prolonged 23% (P<0.05). Significantly greater effects were observed in subendocardial myocytes, in which a phase 2 shoulder began to appear (Figure 2B), and the APD increased 60% (P<0.01) at 3 weeks (Figure 2C). Comparison of subepicardial and subendocardial responses to hemodynamic stress (Figure 2D) revealed a strikingly greater degree of action potential prolongation (measured as APD₉₀ or APD₅₀) in subendocardial cells (P<0.01). Current- and voltage-clamp recordings in sham-operated mice were similar to those of unoperated controls.

View larger version (26K): [in this window] [in a new window]   Figure 2. Representative action potentials (stimulation cycle length, 200 ms) recorded from isolated ventricular myocytes (A, subepicardial fraction; B, subendocardial fraction) from hearts isolated under sham-operated (A), TAB+CsA (B), or TAB+Veh (C) conditions. C, APD90 (±SEM) measured in subepicardial (subepi) and subendocardial (subendo) myocytes from hearts treated as listed. D, Percent change in APD90 and APD50 between hypertrophied myocytes (TAB+Veh) and control (Sham+Veh). *P<0.05, **P<0.01.

Calcineurin inhibition completely blocked the increases in cell size associated with TAB (Table 2). Despite elimination of the structural changes of hypertrophy, APD prolongation was blunted though not normalized by calcineurin inhibition (Figure 2, Table 1), suggesting that the structural and electrophysiological components of remodeling in hypertrophy are partially dissociable.

Repolarizing K⁺ Currents
I_to is a major determinant of repolarization in murine ventricular myocytes and is frequently implicated in the electrical remodeling of heart failure.¹⁷ We studied the effects of TAB+Veh and TAB+CsA on I_to and on the current that persists at 300 ms (I_sus). At baseline, I_to was relatively large in myocytes isolated from the subepicardial aspect of the LV (Figure 3A). With hypertrophy, I_to and I_sus did not change significantly (Figure 3B, Table 1), and the proportion of subendocardial cells exhibiting substantial I_to (32/43 cells from 16 mice, 74%) was unchanged. Inhibition of hypertrophy with calcineurin blockade (TAB+CsA) was associated with similar I_to and I_sus waveforms (Figure 3C) and densities (Figures 3E and 3F), as was exposure to CsA alone (Figures 3D through 3F, Table 1). Calcineurin inhibition (TAB+CsA) did not alter the proportion of subepicardial myocytes exhibiting I_to (17/27 cells from 10 mice, 63%). I_to and I_sus densities were similar under conditions of TAB+CsA and Sham+CsA (Figures 3E through 3F, Table 1).

View larger version (32K): [in this window] [in a new window]   Figure 3. Whole-cell voltage-clamp K+ currents recorded from LV myocytes (subepicardial fraction) isolated from hearts subjected to Sham+Veh (A), TAB+Veh (B), TAB+CsA (C), and Sham+CsA (D) conditions. Current density–voltage relations for Ipeak (E) and Isus (F) recorded from subepicardial myocytes isolated from hearts treated as listed.

In contrast with subepicardial cells, LV myocytes isolated from the subendocardial fraction exhibited relatively little I_to at baseline (see online Figure IA at www.circulationaha.org). Similar to subepicardial cells, hypertrophy (TAB+Veh) had no effect on I_to or I_sus waveforms (online Figure IB) and densities (online Figures IE through IF, Table 1). The proportion of cells exhibiting substantial I_to was not altered by hypertrophy (TAB+Veh, 12/36 cells from 13 mice, 33%) or by exposure to CsA (TAB+CsA, 10/22 cells from 9 mice, 45%). I_to and I_sus densities were similar under conditions of TAB+CsA and Sham+CsA (online Figures IE and IF, Table 1).

Inward rectifier current (I_K1) densities and kinetics did not differ between subepicardial and subendocardial cells. I_K1 recorded from hypertrophied myocytes (TAB+Veh) was similar to baseline (control and sham operated) and after exposure to CsA (TAB+CsA, Sham+CsA; negative data not shown).

Hypertrophic Upregulation of Inward Ca²⁺ Current
Because intracellular Ca²⁺ is increasingly viewed as a central point of regulation in the pathogenesis of hypertrophy¹⁸ and disease-related electrical remodeling,^19,20 we measured the L-type Ca²⁺ current (I_Ca,L) in the presence (TAB+Veh) and absence (Sham+Veh, TAB+CsA, Sham+CsA) of hypertrophy. No differences in I_Ca,L amplitude or kinetics were observed between subendocardial (Figure 4A) and subepicardial (online Figure II) cell fractions. The amplitude of I_Ca,L in hypertrophied myocytes (TAB+Veh), however, was larger than in sham-operated controls (Figure 4B). Normalization to cell size (capacitance) revealed that peak I_Ca,L density increased 26% (P<0.05) in TAB+Veh myocytes (14±1 pA/pF) compared with Sham+Veh cells (11.1±0.4 pA/pF). I_Ca,L density was increased in TAB+Veh cells at all potentials ranging from -20 to +50 mV (Figure 4F).

View larger version (27K): [in this window] [in a new window]   Figure 4. A through D, Representative recordings of ICa,L elicited by 300-ms depolarizations to -20, 0, +20, and +40 mV from holding potential of -50 mV. E, Voltage protocol used for recording current-voltage relations. F. ICa,L-voltage curves from Sham+Veh (, n=22/6), TAB+Veh (•, n=18/6), TAB+CsA (, n=15/5), and Sham+CsA (, n=15/5) LV.

Peak I_Ca,L and current-voltage relations in cells exposed to TAB+CsA (Figure 4C) were not significantly different from sham-operated controls. The peak I_Ca,L density measured in Sham+CsA cells was slightly less than that measured in Sham+Veh myocytes (9.3±0.9 pA/pF, P<0.05).

To determine whether changes in channel availability underlie the differences in peak I_Ca,L, we examined the voltage dependence of activation and inactivation (Figures 5A through 5C). This analysis revealed no significant differences in steady-state voltage-dependent activation or inactivation of I_Ca,L among any of the 4 treatment groups (Figure 5C). Because intracellular Ca²⁺, acting through calmodulin,²¹ modulates the kinetics of L-type Ca²⁺ channel inactivation, we examined the time dependence of recovery from steady-state I_Ca,L inactivation by using a paired-pulse protocol (Figure 5D). The time constants of recovery from inactivation for TAB+Veh and Sham+Veh cells were 65±7 ms (²=0.0055) and 168±11 ms (²=0.0067), respectively (P<0.05), revealing a significant acceleration of recovery from inactivation in hypertrophied myocytes. The faster recovery from I_Ca,L inactivation was absent in nonhypertrophied banded hearts (TAB+CsA, Figure 5F). The time constants of recovery from steady-state inactivation in TAB+CsA (149±14 ms, ²=0.00033) and Sham+CsA (130±13 ms, ²=0.00031) cells were not significantly different from sham-operated controls.

View larger version (51K): [in this window] [in a new window]   Figure 5. Voltage-dependent ICa,L activation, inactivation, and recovery from inactivation. B, Typical recording of ICa,L elicited by protocol shown in A and C. Mean voltage-dependent activation and inactivation curves for Sham+Veh (, n=11/5), TAB+Veh (•, n=8/6), TAB+CsA (, n=8/4), and Sham+CsA (, n=8/4) LV. Data points were fitted to Boltzmann functions, where V0.5 (d) and V0.5(f) were +2.6±0.4 and -10±1 mV (Sham+Veh), +2.1±0.5 and -10±2 mV (TAB+Veh), +3.2±0.4 and -8.7±1 mV (TAB+CsA), and +3.4±0.6 and -8.3±2 mV (Sham+CsA), respectively. Fits were based on maximally available current densities as follows (pA/pF): Sham+Veh, 11.2±0.6; TAB+Veh, 14.0±0.8; TAB+CsA, 10±1; and Sham+CsA, 8±1. E, Typical recording of ICa,L elicited by protocol shown in D. F, Time course of recovery from inactivation of ICa,L for Sham+Veh (, n=10/5), TAB+Veh (•, n=10/4), TAB+CsA (, n=8/4) and Sham+CsA (, n=6/3) LV. Each curve was adequately fit by single exponential function.

	Discussion

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In recent years, electrophysiological remodeling has emerged as an important pathophysiological mechanism in many types of cardiac pathology. Because clinical heart disease often involves both hypertrophic and failure cellular phenotypes, we sought to elucidate the contribution of hypertrophy in isolation. Working with a model of pressure overload that induces cardiac hypertrophy without LV dysfunction, we evaluated action potential characteristics across the LV and studied the major currents involved in repolarization. We identified differences within the LV in cellular APD that are amplified by hemodynamic stress, and we addressed the role of a recently described calcineurin signaling pathway.

Accumulating evidence points to disorders of ventricular refractoriness underlying ventricular tachyarrhythmias.³ Data from clinical electrophysiological studies, surface electrocardiography, and animal models all point to dispersion of ventricular refractoriness as a major predisposing factor in the development of reentrant ventricular arrhythmias.^22,23 At a basic level, it is becoming increasingly apparent that the electrophysiological characteristics of ventricular tissue are heterogeneous across the ventricular wall,^9,24,25 including murine ventricle,^14,26 and that these regional differences are altered in hypertrophy.^27,28 It will be important in the future to determine why myocytes from different regions of the LV wall (1 mm thick in mouse heart) display markedly different electrophysiological phenotypes.

The cellular and molecular phenotype of hypertrophy is modulated by the degree of hypertrophy and the primary lesion that induces hypertrophy.²⁹ In the TAB hypertrophy model, outward K⁺ currents (I_to, I_K1) do not change, whereas the inward current (I_Ca,L) increases, thus inducing greater APD prolongation in subendocardial myocytes, given their relative lack of repolarizing I_to. Other models of moderate hypertrophy also manifest increased I_Ca,L.^19,30 These findings contrast with numerous models of heart failure in which outward K⁺ currents, especially I_to, are decreased.^19,30–32 Differences in mechanisms underlying action potential prolongation in hypertrophy and heart failure may have significant implications in devising therapeutic approaches to treating arrhythmias in these diseases.

TAB in mice induces 50% cardiac hypertrophy at 3 to 5 weeks, and this process is blocked by calcineurin inhibition (see Table 2 and Hill et al¹³). We have exploited calcineurin inhibition with CsA to control for the structural changes of hypertrophic growth. Calcineurin blockade eliminated the increases in heart mass and myocyte size associated with pressure overload and attenuated the APD prolongation of hypertrophy in both subepicardial and subendocardial myocytes. Similarly, calcineurin blockade prevented the upregulation of I_Ca,L and prevented the accelerated recovery from I_Ca,L inactivation seen with hypertrophy. At present, we do not know whether the CsA-mediated changes in I_Ca,L are secondary to the abrogated structural changes of hypertrophy, vice versa, or are separate phenomena. Because the action potential prolongation did not normalize completely with CsA, electrogenic transport processes in addition to I_Ca,L must be involved. Nevertheless, it is possible that the modulatory effects of CsA on hypertrophy are mediated via the L-type channel; a model could be proposed wherein CsA decreases I_Ca,L, which diminishes intracellular Ca²⁺, a central point in several hypertrophic signaling pathways.^18,33

Calcineurin was inhibited with CsA at a dose that produced CsA trough levels of 2.6±0.2 µg/mL,¹³ which inhibited 90% of total calcineurin phosphatase activity in the heart. Our assay of calcineurin activity relies on myocyte disruption with provision of exogenous Ca²⁺ and calmodulin, thereby activating total myocyte stores of calcineurin. Although documenting the efficacy of calcineurin inhibition by CsA, this assay does not distinguish between activated and inactive calcineurin within the cell.

The central role of intracellular calcium signaling in hypertrophy is established. In the heart, a small amount of Ca²⁺ entering through L-type channels triggers the release of much larger amounts of Ca²⁺ from intracellular stores. Thus, modest changes in Ca²⁺ influx are amplified within the cell. We predict that hypertrophy-associated I_Ca,L upregulation may generate positive feedback in the hypertrophic cascade. CsA-sensitive changes in I_Ca,L inactivation kinetics are suggestive of calcineurin-mediated regulation of the Ca²⁺ channel complex, as has been reported for a number of proteins involved in Ca²⁺ homeostasis, including the 1,4,5-triphosphate and ryanodine receptors, and the neuronal and cardiac³⁴ Na⁺-Ca²⁺ exchangers (reviewed in Carafoli et al³⁵).

Cardiac hypertrophy has been viewed as a pathological milestone in disease progression leading to heart failure, a syndrome in which compensatory responses such as vasoconstriction, neurohumoral recruitment, and cytokine activation are maladaptive in the long term. To this list, we must add I_Ca,L upregulation and action potential prolongation.^19,32 Although an increased influx of Ca²⁺ may prove beneficial by maximizing inotropic potential in the short run, the resulting action potential prolongation heightens dispersion of refractoriness and predisposes to afterdepolarizations and triggered automaticity, reentry, and arrhythmogenesis.

	Acknowledgments

 
This work was supported by grants from the Donald W. Reynolds Cardiovascular Clinical Research Center, American Heart Association–Heartland Affiliate, Department of Veterans Affairs, National Institutes of Health grant HL-03908, and the Roy J. Carver Charitable Trust. We sincerely thank Drs Donald Heistad, Michael Welsh, and Kevin Campbell for helpful advice and support.

	Footnotes

 
Figures I and II can be found Online at http://www.circulationaha.org

Received February 12, 2001; revision received June 21, 2001; accepted June 22, 2001.

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