Cardiovascular Research

Cardiovascular Research 1998 37(2):432-444; doi:10.1016/S0008-6363(97)00128-4
© 1998 by European Society of Cardiology

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Copyright © 1998, European Society of Cardiology

Changes in L-type calcium channel abundance and function during the transition to pacing-induced congestive heart failure

Rupak Mukherjee, Kenneth W Hewett, Jennifer D Walker, Charles G Basler and Francis G Spinale^*

Divisions of Cardiothoracic Surgery and Pediatric Cardiology, Medical University of South Carolina, Charleston, SC, USA

^* Corresponding author. Division of Cardiothoracic Surgery, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA. Tel.: +1 (803) 792-2011; fax +1 (803) 792-8286.

Received 8 November 1996; accepted 27 March 1997

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: The development of congestive heart failure (CHF) is accompanied by left ventricular (LV) and myocyte contractile dysfunction. However, time-dependent cellular and ionic events which contribute to the initiation and progression of CHF remain unclear. This study tested the central hypothesis that changes in L-type Ca²⁺ channel current (I_Ca) and abundance (B_max) are early events in the transition to CHF. Methods: LV fractional shortening by echocardiography, isolated LV myocyte shortening velocity by videomicroscopy, I_Ca by voltage-clamp, and B_max by [³H]nitrendipine binding were determined at each week during the progression of pacing-induced CHF in pigs (240 bpm; n=6/week for 3 weeks). Myocyte and L-type Ca²⁺ channel function were determined under basal conditions and after β-adrenergic receptor stimulation with 25 nM isoproterenol. Results: After 1 week of pacing, myocyte and L-type Ca²⁺ current responses to β-adrenergic receptor stimulation were reduced by 20% from control values and was accompanied by over a 210% increase in plasma catecholamine levels. After 2 weeks of pacing, reductions in LV fractional shortening and myocyte shortening velocity from control values (20±1 vs. 34±2% and 36.7±2.9 vs. 50.6±2.4 µm/s, respectively, P<0.05) were paralleled by decreased I_Ca (2.47±0.10 vs. 3.63±0.25 pA/pF, P<0.02) and B_max (149±16 vs. 180±12 fmol/mg, P<0.03). After 3 weeks of pacing, LV fractional shortening was reduced by over 50%, myocyte shortening velocity by 37%, and I_Ca and B_max were reduced by over 25% from control values. Furthermore, after 3 weeks of pacing, the I_Ca/B_max ratio was reduced from control values (16.2±0.9 vs. 20.6±1.2 [fA/pF]/[fmol/mg], P<0.03), which suggests functional defects in the remaining L-type Ca²⁺ channels. Conclusions: An early event during the transition to pacing-induced CHF was diminished β-adrenergic receptor augmented L-type Ca²⁺ current, which was followed by an absolute loss of steady-state L-type Ca²⁺ current and channel abundance. The development of severe CHF was accompanied by a loss of Ca²⁺ carrying capacity through residual channels. These unique findings suggest that a contributory molecular mechanism for the initiation and progression of CHF is changes in the structure and function of the L-type Ca²⁺ channels.

KEYWORDS Calcium channel, L-type; Heart failure; Echocardiography; Isoproterenol; Pig, ventricular myocytes

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Congestive heart failure (CHF) is a significant cause of morbidity and mortality worldwide and is characterized by reduced left ventricular (LV) pump function and neurohormonal system activation [1–4]. An animal model of pacing-induced CHF has been employed previously in order to examine functional and neurohormonal events which occur with the initiation and progression of CHF [5–22]. Specifically, this laboratory has demonstrated that the development of pacing-induced CHF in pigs resulted in isolated myocyte contractile dysfunction [13, 18–22]. An important ionic event which initiates the contractile process is activation of the voltage-sensitive L-type Ca²⁺ channel. Specifically, Ca²⁺ influx through activated L-type Ca²⁺ channels triggers Ca²⁺ release from sarcoplasmic reticular stores, and the resultant increase in cytosolic free Ca²⁺ leads to actin–myosin crossbridge formation [23, 24]. This laboratory has recently demonstrated that a significant reduction in the L-type Ca²⁺ current occurred with the development of pacing-induced CHF and was associated with myocyte contractile dysfunction [18]. However, it remained unknown from these studies whether defects in L-type Ca²⁺ channel function were an early or late event in the progression of this pathological process. Accordingly, the overall goal of this project was to examine the temporal relationship between LV pump function, isolated myocyte contractile function, and L-type Ca²⁺ channel function during the progression of pacing-induced CHF.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Animal model of pacing-induced CHF
Congestive heart failure (CHF) was induced in pigs (Yorkshire strain, neutered males, 6 months, 25–28 kg; Hambone Farms, Orangeburg, SC) by chronic supraventricular pacing using techniques well described by this laboratory [18–21]. Briefly, platinum electrodes were sutured onto the left atrium and modified pacemakers were buried in a subcutaneous pocket. Following recovery from the surgical procedure, atrial pacing at 240 bpm was initiated. A total of 24 pigs were used for this study. Six pigs each were studied after 1, 2, and 3 weeks of supraventricular pacing as outlined above. Six pigs served as sham-operated controls. Echocardiograms and electrocardiograms were obtained on a weekly basis during the pacing protocol in order to ensure the presence of 1:1 electrical conduction and electromechanical coupling. All animals were treated and cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals [25].

2.2 LV function and plasma catecholamines with pacing-induced CHF
At the end of the pacing protocol, LV function was assessed using echocardiography by methods well described [18–21]. On the day of study, animals were sedated with 10 mg of midazolam, placed in a custom-designed sling which allowed the animal to rest comfortably, an ECG was established, and the pacemakers deactivated (paced groups only). After a 30 min stabilization period, 10 ml of blood was drawn from the precava into tubes containing EDTA (1.5 mg/ml) and was used for the determination of plasma catecholamines. Two-dimensional and M-mode echocardiographic studies were used to image the LV from a right parasternal approach. LV fractional shortening was computed as: [(end-diastolic dimension – end-systolic dimension)/end-diastolic dimension] and expressed as a percentage. Following LV function measurements the animal was anesthetized with isoflurane (2.0%/1.5 l/min) and nitrous oxide (0.5 l/min), a sternotomy was performed, the heart quickly extirpated and placed in an oxygenated Krebs' solution. The region of the LV free wall comprising the left circumflex coronary artery was dissected free, the artery cannulated, and prepared for myocyte isolation. The region of the LV free wall comprising the left anterior descending artery and the posterior portion of the LV free wall were quickly rinsed free of blood, epicardial fat trimmed away, and rapidly frozen in liquid nitrogen for biochemical and molecular assays.

For plasma catecholamine determination, blood samples collected at the time of final study were immediately centrifuged (2000xg, 10 min, 4°C), the plasma decanted into separate tubes, frozen in liquid nitrogen, and stored at –80°C until the time of assay. The plasma samples were assayed for catecholamine concentration by radioenzymatic assay (TRK 995, Amersham, Arlington Heights, IL).

2.3 Myocyte isolation and myocyte contractile function
Using methods developed by this laboratory [9, 13, 18–21], LV myocytes were isolated using a collagenase solution (0.5 mg/ml). The tissue was then minced into 2 mm sections and added to an oxygenated cell-dispersion solution containing 400 µM CaCl₂ and collagenase. The liberated myocytes were resuspended in cell medium (Medium 199; 2 mM Ca²⁺). Isolated myocytes were placed in a thermostatically controlled chamber (2.5 ml, 37°C) fitted with a coverslip on the bottom for imaging on an inverted microscope. Myocyte contractions were elicited by electrical field stimulation at 1 Hz (S11, Grass Instruments, Quincy, MA) and imaged using a charge-coupled device with a non-interlaced scan rate of 240 Hz (GPCD60, Panasonic, Secaucus, NJ). This video signal input through an edge detector system (Crescent Electronics, Sandy, UT), and digitized (ATMIO16, National Instruments, Austin, TX). Stimulated myocytes were allowed a 5 min stabilization period following which contraction data for each myocyte were recorded from a minimum of 20 consecutive contractions. Parameters computed from the digitized contraction profiles included shortening extent (µm) and peak velocity of shortening (µm/s). Following collection of baseline indices of myocyte function, 25 nM (–) isoproterenol was added to the tissue chamber and the contractile response of the myocytes was recorded a minimum of 5 min after the addition. This concentration of isoproterenol has been demonstrated previously to elicit a near-maximal contractile response for this myocyte preparation [13].

2.4 L-type Ca²⁺ currents
Whole-cell L-type Ca²⁺ currents were measured using methods described by this laboratory previously [18]. Briefly, isolated myocytes were placed in a 0.5 ml lucite tissue chamber mounted on the stage of an inverted microscope and superfused with a modified Krebs' solution containing (mM): NaCl 133, KCl 4.7, dextrose 16.5, HEPES 20, MgCl₂ 1.2, and CaCl₂ 2.5 (pH 7.44). Microelectrodes (tip resistance: 1.5–4 M) were heat-polished before use and filled with a solution containing (mM): CsOH 110, aspartic acid 90, CsCl 20, tetraethylammonium chloride 10, HEPES 5, ATP (Mg²⁺ salt) 5, EGTA 10, and Na₂-creatine phosphate 5 (pH 7.2; 290 mOsm) by immersing the tips in the pipette solution, then backfilled with pipette solution containing 100 µg/ml nystatin [26]. Following the formation of a gigaohm seal, electrical access to the cell interior was assessed by the change in capacitance measured at the microelectrode tip [18, 26]. When the patch was sufficiently permeabilized by nystatin, the superfusate solution was changed to a Na⁺- and K⁺-free solution that contained the following (mM): choline chloride 145, dextrose 5.5, MgCl₂ 1.2, HEPES 5, CaCl₂ 2.5, and 4-aminopyridine 2 (pH 7.2). All L-type Ca²⁺ channel currents were measured at room temperature (22–23°C) in order to enhance the temporal resolution of peak L-type Ca²⁺ current measurements. Following the measurement of L-type Ca²⁺ currents under steady-state (baseline) conditions, 25 nM (–)-isoproterenol [13]was added to the tissue chamber and L-type Ca²⁺ currents were recorded 5 min following the addition.

2.4.1 Voltage clamp protocols
Membrane voltage clamping protocols were generated using the pClamp software (version 6.0; Axon Instruments, Burlingame, CA) and a 12-bit digital to analog convertor (LabMaster TL-1; Axon Instruments, Burlingame, CA).

2.4.1.1 Measurement of membrane capacitance
As described previously [18], a voltage ramp protocol was employed to measure the membrane capacitance. Briefly, a voltage ramp of 2.14 V/s was initiated from a holding potential of –100 mV. The membrane capacitance was calculated from the height of the current step using the relationship:

where C_m is the membrane capacitance and dV/dt is the rate of the voltage ramp. In preliminary experiments, values for membrane capacitance obtained by using the ramp technique were similar to the those measured using the time constant of inward current inactivation following membrane hyperpolarization from –80 to –100 mV [27].

2.4.1.2 Current vs. voltage relationship
In order to examine the current–voltage (I–V) relationship for the L-type Ca²⁺ channels, the membrane holding potential was –40 mV [18, 26]. Clamp steps from –50 to +60 mV and 250 ms in duration were applied at 5 s intervals. All inward currents were normalized to membrane capacitance.

2.4.1.3 Steady-state inactivation measurements
Steady-state inactivation characteristics for the L-type Ca²⁺ current were examined using a two-pulse protocol as previously described [18, 26–29]. Briefly, L-type Ca²⁺ currents were recorded by preceding a 250 ms step in the command voltage from –40 to +20 mV by a 500 ms conditioning (V_cond) step. Depolarizing V_cond steps were increased from –50 to +25 mV in 5 mV increments. A 2 ms interval was introduced between the conditioning and test pulses during which the membrane was held at –40 mV. This stimulation pattern was repeated at an interval of 5 s.

2.4.1.4 Data recording and determinants of L-type Ca²⁺ currents
Current signals were low-pass filtered at 5 kHz and amplified (Axopatch 200A; Axon Instruments, Burlingame, CA). Parameters computed from the current–voltage relationship for the L-type Ca²⁺ channels included the maximum inward current, and the membrane potential at which the Ca²⁺ channel current flow was maximal. Peak inward current (I_Ca) was measured as the difference between the maximal current at activation and the steady-state current at the end of the voltage clamp step [26–29]. The apparent reversal potential (V_rev), which is defined as the membrane potential at which the Ca²⁺ current changes from an inward to an outward flux of Ca²⁺ ions [28, 29], was computed by extrapolating the ascending limb of the current–voltage relationship to intercept the voltage-axis. In order to examine the voltage-dependence of activation (d), conductance (G) was computed using the equation [29]:

(1)

where V_m is the membrane voltage of the step pulse.

Values obtained for G were normalized to the maximal conductance (G_max) and fitted to the Boltzmann function [29]given by:

(2)

where V_0.5 is defined as the membrane potential at which 50% of the L-type Ca²⁺ channels would be available for conduction and k, the slope factor, is defined as the voltage-dependent rate of availability of the Ca²⁺ channels.

Parameters computed from the steady-state inactivation (f) characteristics for the L-type Ca²⁺ currents included the membrane voltage at which 50% of the channels were inactivated (V_0.5) and the voltage-dependent rate of inactivation (slope factor, k) [18, 28]. Peak I_Ca recorded during the test pulse (I_t) were normalized to the maximal current recorded without a conditioning pulse (I_max) and fitted to a Boltzmann function [18, 28]given by:

(3)

where V_cond was the voltage of the conditioning step.

2.5 L-type Ca²⁺ channel abundance
Isolated membrane preparations were used to determine the abundance of the L-type Ca²⁺ channels. Membrane preparations were obtained from the LV myocardium employing techniques described by this laboratory previously [19]. Briefly, 15 g of LV free wall, from which the epicardial fat had been trimmed away, was placed in 10 volumes of ice-cold buffer containing 250 mM sucrose, 5 mM Tris, and 1 mM EGTA and homogenized. The homogenate was centrifuged at 250xg for 10 min, the pellet discarded, and the supernatant spun at 50 000xg for 15 min. The resultant pellet was resuspended with an ice-cold buffer of 50 mM Tris-HCl (pH 7.4). The preparation was recentrifuged and resuspended twice in Tris buffer. In order to ensure that membrane protein was not lost during centrifugation, supernatants from each step of the isolation procedure were examined for Na⁺,K⁺-ATPase activity by assaying for p-nitrophenophosphatase activity [19]. The final concentration of protein in the membrane preparations was 8.9±1.2 mg/g of LV myocardium with an approximately 20-fold enrichment, as assessed by an increase in p-nitrophenophosphatase activity. A consistent membrane yield was obtained for all LV myocardial preparations.

Binding experiments were performed using radiolabelled dihydropyridines [15, 30]. Specifically, dihydropyridine binding was performed on membrane preparations using [³H]nitrendipine as described previously [30]. Briefly, membrane preparations (0.08–0.1 mg protein/tube) were incubated with 0.25–10 nM [³H]nitrendipine in the absence (total binding) or presence (non-specific binding) of 100 µM unlabelled nifedipine. This concentration of nifedipine has been previously demonstrated to inhibit more than 95% of the specific [³H]nitrendipine binding [30]. The reaction volume for this assay was 250 µl. Samples were incubated in the dark for 60 min after which the reactions were terminated by the addition of 1 ml ice-cold Tris-HCl buffer and vacuum filtration through Whatman GF/C filters. The filters were placed in vials containing 10 ml scintillation fluid and the radioactivity counted on a scintillation counter at an efficiency of 39–41%. All binding assays were performed in duplicate for each pig, and specific binding was determined by subtracting non-specific binding from total binding. Maximal binding (B_max) and dissociation constant (K_d) values were determined by Scatchard analysis [30].

2.6 Data analysis
Indices of LV pump function, myocyte contractile function, and L-type Ca²⁺ channel function with increasing durations of pacing were compared using analysis of variance. For the analyses of myocyte and L-type Ca²⁺ channel function, each pig was considered a complete block. Thus, the numbers of myocytes studied from each pig were considered as repeated observations within each block. If the analysis of variance revealed significant differences, pairwise tests of individual group means were compared using Bonferroni probabilities [31]. Results from the dihydropyridine binding studies were compared using analysis of variance and mean separation was performed in a similar fashion. Linear and non-linear regression analyses to determine the apparent reversal potential as well as activation and inactivation characteristics of the L-type Ca²⁺ current were performed using regression modules provided with the BMDP statistical software package. All statistical procedures were performed using the BMDP statistical software package (BMDP Statistical Software Inc., Los Angeles, CA). Results are presented as mean±standard error of the mean (s.e.m.). Values of P<0.05 were considered to be statistically significant.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Pigs were successfully studied at 1, 2, and 3 weeks after the initiation of rapid atrial pacing. The animals following 3 weeks of rapid pacing exhibited signs and symptoms of CHF which included dyspnea, ascites, and peripheral edema.

3.1 LV geometry and pump function
LV geometry and pump function measurements were obtained in the conscious state and at an ambient resting heart rate (pacemakers deactivated). The resting heart rate was 115±8 bpm in the control group and did not change significantly until 3 weeks of rapid pacing (143±9 bpm; P<0.05). Weekly changes in LV end-diastolic dimension, LV fractional shortening, and plasma catecholamine concentrations with rapid pacing are presented in Fig. 1. LV end-diastolic dimension was significantly increased from control values with 1 week of rapid pacing and remained elevated with longer pacing durations. LV fractional shortening was reduced at 1 week of pacing (P=0.11) and significantly declined from control values following 2 weeks of rapid pacing. Plasma catecholamine concentrations were significantly elevated by 1 week of rapid pacing and remained elevated through the pacing protocol. Thus, the transition to CHF induced by rapid pacing was associated with progressive LV pump dysfunction and neurohormonal system activation.

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Weekly changes in left ventricular (LV) end-diastolic dimension, LV fractional shortening, and plasma catecholamine concentrations with chronic rapid pacing. LV chamber dimension was significantly increased by 1 week of pacing and continued to increase with longer durations of pacing. A decline in LV fractional shortening was seen at 1 week of pacing, but did not reach statistical significance (P=0.11). After 2 weeks of rapid pacing, LV fractional shortening was significantly reduced from control values and decreased further at 3 weeks of pacing. Plasma catecholamine concentrations rose significantly from control values by 1 week of rapid pacing and remained persistently elevated with longer pacing durations. *P<0.05 vs. control; +P<0.05 vs. 1 week values; control=time 0. n=6 each group.

 
3.2 Steady-state myocyte contractility
Steady-state myocyte contractile function was measured in an average of 300 cardiocytes under control conditions and with each week of rapid pacing (minimum of 35 myocytes per pig). Values obtained for resting myocyte length with each week of rapid pacing are summarized in Table 1. Myocyte resting length increased by 1 week of rapid pacing and remained elevated with longer pacing durations. Weekly changes in myocyte extent and velocity of shortening with rapid pacing are shown in Fig. 2. A significant reduction in myocyte extent and velocity of shortening from control values was observed after 2 weeks of rapid pacing. Myocyte extent and velocity of shortening progressively declined with longer pacing durations, and were significantly lower at 3 weeks of pacing than 1-week values.

View this table: [in this window] [in a new window]   Table 1 Myocyte length, membrane capacitance, and L-type Ca2+ current characteristics with each week of rapid pacing

 

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Myocyte extent and velocity of shortening were determined in over 300 myocytes in the control group (time 0) and with each week of rapid pacing. For the analysis of myocyte contractile function, each pig was considered to be a single block. All myocytes from each pig were considered as repeated measurements within a single block. A significant and progressive decline in myocyte extent and velocity of shortening values was observed after 2 weeks of rapid pacing. By 3 weeks of pacing, myocyte extent and velocity of shortening were significantly lower than 1 week values. *P<0.05 vs. control; +P<0.05 vs. 1 week values; control=time 0. n=6 each group.

 
3.3 Steady-state L-type Ca²⁺ channel function
Steady-state, whole cell L-type Ca²⁺ channel current characteristics were examined in an average of 27 myocytes from each group (minimum of 4 myocytes per pig). Representative L-type Ca²⁺ current profiles recorded from a control myocyte and myocytes with each week of rapid pacing at specific depolarizing voltage steps are shown in Fig. 3. Peak inward current was normalized to membrane capacitance and plotted as a function of the membrane voltage to generate current–voltage relationships for the myocytes from the control and all the pacing groups (Fig. 4). Values obtained for membrane capacitance and specific indices of L-type Ca²⁺ current activation with each week of rapid pacing are summarized in Table 1. A significant reduction in the peak L-type Ca²⁺ current from control and 1 week values was observed following 2 weeks of pacing. There were no significant differences in the membrane voltage at peak current, or the apparent reversal potential between the control and the rapid pacing groups. Membrane capacitance was increased by 1 week of rapid pacing and remained elevated with longer pacing durations. This increase in membrane capacitance (or cell surface area) may have been due to changes in myocyte geometry (e.g., resting myocyte length) that occurred with increased durations of rapid pacing. In light of the fact that changes in membrane capacitance may have influenced L-type Ca²⁺ current measurements, peak L-type Ca²⁺ currents were examined in a subset of myocytes from the control group and each week of pacing (n=5) which had similar membrane capacitances (Fig. 5). Membrane capacitance from this subset of control myocytes was 121±3 pF and was not significantly different in any of the rapid pacing groups (1 week: 131±4; 2 weeks: 124±2; 3 weeks: 126±3; P>0.12). As shown in Fig. 5, in myocytes with equivalent membrane capacitances, a significant reduction in peak L-type Ca²⁺ current from control values was observed after 2 weeks of rapid pacing.

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Representative L-type Ca2+ current recordings from a control myocyte and myocytes with each week of rapid pacing at baseline and from the same myocytes in the presence of isoproterenol. L-type Ca2+ currents were recorded at depolarizing voltages of –50 to +60 mV from a holding potential of –40 mV. This figure shows representative current traces recorded at depolarizing voltages of –35, –10, 0, and +10 mV. The membrane capacitances of the myocytes were: control, 108 pF; 1 week, 147 pF; 2 weeks, 133 pF; 3 weeks, 136 pF. In the presence of isoproterenol, the magnitude of the inward current was increased from baseline values in the control as well as all the pacing groups. Specific indices of L-type Ca2+ current characteristics are summarized in Table 1. Dashed lines indicate zero current.

 

View larger version (25K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Current–voltage relationships for the L-type Ca2+ current obtained from a minimum of 24 myocytes in the control (time 0) group and all the rapid pacing groups. With 1 week of rapid pacing, there was no change in the peak L-type Ca2+ current. With longer durations of pacing, a significant reduction in the peak L-type Ca2+ current occurred. Values obtained for peak L-type Ca2+ current and the membrane potential at which peak current was recorded are summarized in Table 1.

 

View larger version (10K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Peak L-type Ca2+ current recorded in a subset of control myocytes and myocytes with each week of rapid pacing which had similar membrane capacitances (n=5 myocytes each group). The membrane capacitance for the control myocytes was 121±3 pF and was not significantly different in any of the pacing groups. With 1 week of rapid pacing, there was no change in the peak L-type Ca2+ current. With longer durations of pacing, a significant reduction in the peak L-type Ca2+ current occurred. *P<0.05 vs. control; +P<0.05 vs. 1 week values; control=time 0.

 
The voltage-dependence of activation and inactivation for the L-type Ca²⁺ channels was determined in order to examine whether the progression of pacing-induced CHF had altered these parameters. Boltzmann functions were fitted to the steady-state activation and inactivation data in order to determine the voltage at which 50% of the channels would be activated (Eq. 2) or inactivated (Eq. (3); V_0.5) and the slope factors (k). Specific values obtained for these parameters are summarized in Table 1. There were no significant differences in V_0.5 or k between the control and any of the pacing groups. In light of the fact that the voltage-dependent activation and inactivation characteristics of the L-type Ca²⁺ currents were unaffected with the progression of pacing-induced CHF, it is likely that the reduction in the peak L-type Ca²⁺ current observed in the present study may have been due to a reduction in the current carrying capacity of the L-type Ca²⁺ channels.

3.4 Myocyte and L-type Ca²⁺ channel function with β-adrenergic receptor stimulation
In the next series of experiments, the effects of β-adrenergic stimulation on myocyte contractile function and peak L-type Ca²⁺ currents with the progression of pacing-induced CHF were examined. As shown in Fig. 3, there was an observable increase in the peak L-type Ca²⁺ current from baseline values in the presence of isoproterenol. Changes in L-type Ca²⁺ current characteristics with β-adrenergic stimulation are summarized in Table 1. In the presence of isoproterenol, peak L-type Ca²⁺ current was increased from baseline values in the control and all the pacing groups. However, peak L-type Ca²⁺ current with β-adrenergic stimulation was lower in all the pacing groups than in controls. In light of the differences in basal myocyte and L-type Ca²⁺ channel function in the control and the chronic pacing groups, the magnitude of the response to β-adrenergic receptor stimulation was examined as the absolute increase in shortening velocity and peak L-type Ca²⁺ current, respectively. The results from these analyses are summarized in Fig. 6. A significant fall in myocyte contractile responsiveness to β-adrenergic receptor stimulation occurred after 1 week of rapid pacing and remained lower with longer durations of pacing. Compared to 1 week values, a significant reduction in myocyte β-adrenergic responsiveness had occurred with 3 weeks of rapid pacing. With β-adrenergic receptor stimulation, peak L-type Ca²⁺ current was reduced by 1 week of pacing and remained lower than control values with longer durations of pacing. While the voltage at which 50% of the L-type Ca²⁺ channels were activated (V_0.5 from the d curve) were significantly lower than baseline values, this parameter was similarly reduced in the control and all the rapid pacing groups (Table 1). There were no significant differences in the membrane voltage at peak current, the apparent reversal potential, and steady-state inactivation characteristics either from baseline values or between the control and the rapid pacing groups (Table 1).

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Response of myocyte contractile function and L-type Ca2+ currents to β-adrenergic receptor stimulation with isoproterenol. In light of the fact that differences in baseline measurements of myocyte contractility and L-type Ca2+ currents existed between the control and rapid pacing groups, myocyte and L-type Ca2+ channel function with β-adrenergic receptor stimulation was computed as the change in myocyte shortening velocity and peak L-type Ca2+ current from baseline values, respectively. With 1 week of pacing, myocyte shortening velocity with β-adrenergic receptor stimulation was significantly reduced from control values and remained persistently reduced with longer pacing durations. Similarly, there was a substantial fall in the response of the L-type Ca2+ current with β-adrenergic stimulation by 1 week of pacing which persisted with longer pacing durations. *P<0.05 vs. control; +P<0.05 vs. 1 week values; control=time 0. n=6 each group.

 
3.5 Abundance of L-type Ca²⁺ channels
Specific binding of the dihydropyridine, nitrendipine, was employed to determine the relative abundance of the L-type Ca²⁺ channels. Specific sarcolemmal binding of [³H]nitrendipine was saturable for all the preparations used (Fig. 7). The maximal binding (B_max) for nitrendipine was computed and the results are presented in Fig. 7. Similar to changes in steady-state L-type Ca²⁺ currents with the progression of pacing-induced CHF, a significant decline in B_max occurred after 2 weeks of rapid pacing. The computed dissociation constant for nitrendipine binding was 1.5±0.4 nM in the control group and was unchanged in any of the pacing groups. Thus, the time-dependent reduction in L-type Ca²⁺ current with the progression of pacing-induced CHF was associated with a parallel reduction in the abundance of the L-type Ca²⁺ channel.

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Specific binding of the L-type Ca2+ channel ligand, nitrendipine, to LV sarcolemmal preparations obtained from control (time 0) and with each week of rapid pacing (left panel). Specific binding was saturable in all the sarcolemmal preparations. Scatchard analysis was performed to determine maximal binding (Bmax) and the dissociation constant (Kd) for nitrendipine binding and the results from these analysis are shown in the inset. Weekly changes in Bmax with increasing durations of rapid pacing (right panel) demonstrated a significant reduction from control values after 2 weeks of pacing. Computed values for Kd in the any of the pacing groups were not significantly different from the control group (1.5±0.4 nM). *P<0.05 vs. control; +P<0.05 vs. 1 week values; control=time 0. n=6 each group.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The primary defect in congestive heart failure (CHF) is a reduction in left ventricular (LV) pump function [1, 2, 5]. However, specific cellular and molecular mechanisms which contribute to the progression of this disease process remain unclear. Past studies have shown that chronic rapid pacing in animals cause time-dependent changes in LV function and neurohormonal system activation which are similar to the clinical spectrum of CHF (reviewed in Ref. [5]). This model of rapid pacing was employed in the present study to examine the relationship between the time-dependent changes in LV pump function, isolated myocyte contractile function, and L-type Ca²⁺ channel abundance and function which occurred during the transition to CHF. The new and significant findings from this investigation were 4-fold. First, after 2 weeks of rapid pacing a parallel decline in LV pump function and isolated myocyte contractile function occurred. Second, a contributory mechanism for the defect in LV and myocyte function in this model of CHF was a reduction in the whole-cell L-type Ca²⁺ current. Third, reduced L-type Ca²⁺ current was due, at least in part, to an absolute loss in abundance of the L-type Ca²⁺ channels. Fourth, an early defect with the progression of pacing-induced CHF was a reduction in myocyte and L-type Ca²⁺ current response to β-adrenergic receptor stimulation. Thus, the present study demonstrated for the first time that a temporal relationship exists between changes in a fundamental molecular component of the myocyte excitation–contraction coupling process, the L-type Ca²⁺ current, and myocyte contractility and LV pump function during the transition to pacing-induced CHF. Furthermore, an early event in the progression of pacing-induced CHF was a loss of β-adrenergic-receptor-mediated modulation of L-type Ca²⁺ current and myocyte contractile responsiveness.

A number of past studies have demonstrated that pacing-induced CHF causes progressive LV pump dysfunction and neurohormonal activation [6–11]. For example, Armstrong and colleagues demonstrated that rapid ventricular pacing in dogs caused a progressive increase in LV chamber dimensions and a significant decline in LV ejection performance [6]. In addition, past reports have demonstrated that an early event during the transition to pacing-induced CHF is increased plasma catecholamine concentrations [6–9]. Consistent with the findings of these past reports, results from the present study demonstrated that increased LV chamber dimensions and plasma catecholamine concentrations occurred early during the progression of pacing-induced CHF. The present study builds on these past reports by demonstrating that the cellular basis for the progressive reduction in LV pump performance with chronic pacing was the development of myocyte contractile dysfunction. While reports from this laboratory have demonstrated previously that the development of severe pacing-induced CHF was accompanied by significant defects in myocyte contractile performance [18–22], the results of the present study demonstrate that parallel reductions in myocyte and LV pump function occur during the progression of a CHF process. Results from the present study demonstrated that after 1 week of rapid pacing, basal myocyte contractile function was unchanged, but the capacity of the myocyte to respond to an inotropic stimulus was reduced. Komamura et al. demonstrated that in vivo indices of LV contractile performance were reduced early during the development of pacing-induced CHF [11]. While remaining speculative, results from the present study suggest that a contributory mechanism for the early decline in LV contractile performance with chronic rapid pacing is diminished myocyte response to extracellular stimuli such as neurohormonal activity and changes in loading conditions.

The influx of Ca²⁺ through L-type Ca²⁺ channels is a fundamental event which initiates the myocyte contractile process [23, 24]. Therefore, alterations in L-type Ca²⁺ channel function may adversely affect myocyte contractility. In the present study, 2 weeks of pacing resulted in an over 35% reduction in L-type Ca²⁺ current and myocyte shortening velocity. Thus, likely mechanisms for the LV pump dysfunction which occurred with the progression of pacing-induced CHF include a defect in L-type Ca²⁺ current and myocyte contractile dysfunction. With a longer duration of pacing, this laboratory and others have reported a number of alterations in sarcolemmal transduction and Ca²⁺ homeostatic systems [8, 10, 12–16, 18, 19]. For example, this laboratory has demonstrated previously that significant reductions in the abundance and activity of the Na⁺, K⁺-ATPase system occur with the development of pacing-induced CHF [19]. Cory and colleagues have demonstrated that the development of pacing-induced LV pump dysfunction was associated with reductions in sarcoplasmic reticular Ca²⁺ release and uptake capacities [14]. The present study builds upon these past reports by demonstrating that an early defect during the transition to severe CHF is changes in L-type Ca²⁺ current. In experimental and clinical forms of CHF, a blunted or negative myocardial force–frequency relationship has been reported [17, 21, 32]. For example, Eising et al. demonstrated that with the development of pacing-induced CHF, a significant attenuation in the relation between LV pressure development and stimulation frequency occurred [17]. At the cellular level, this laboratory has reported previously that the development of severe pacing-induced CHF was associated with a significant reduction in the relation between myocyte shortening velocity and stimulation frequency [21]. A past report has demonstrated that the magnitude of the L-type Ca²⁺ current was potentiated with higher stimulation frequencies [33]. Taken together, results from these past reports and the present study suggest that a contributory mechanism for the attenuation of the force–frequency relation which occurs with the development of CHF may be reduced recruitable L-type Ca²⁺ current with increasing stimulation frequencies.

A common means of increasing contractile performance is through β-adrenergic receptor stimulation. One means by which β-adrenergic receptor stimulation increases the inotropic state of the myocyte is to increase the magnitude of the L-type Ca²⁺ current [23, 26, 34, 35]. Past studies have clearly demonstrated an attenuation in myocardial contractile response to β-adrenergic receptor stimulation with the development of CHF [4, 12, 13]. For example, Bristow and colleagues demonstrated that following β-adrenergic receptor stimulation, peak isometric tension was reduced in papillary muscles taken from patients with end-stage heart failure [4]. Previous reports have shown a similar attenuation in the β-adrenergic response of papillary muscles and isolated myocytes following the development of pacing-induced CHF [12, 13]. The present study demonstrated that the transition to pacing-induced CHF was associated with an early decline in myocyte β-adrenergic response and that a contributory mechanism to this effect was reduced L-type Ca²⁺ current augmentation following β-adrenergic receptor stimulation. Normally, β-adrenergic receptor stimulation modulates L-type Ca²⁺ current by causing a cyclic-AMP-dependent phosphorylation of the ₁- and β-subunits of the L-type Ca²⁺ channel [23, 34, 36]. In the present study, an attenuated response of the L-type Ca²⁺ current and myocyte function with β-adrenergic receptor stimulation was observed after 1 week of rapid pacing and was associated with a significant elevation in plasma catecholamine concentrations. Past studies have clearly demonstrated that elevated plasma catecholamine concentrations were associated with desensitization of the β-adrenergic transduction mechanism [3, 4, 8]. This laboratory and others have reported a reduction in cyclic AMP generation with the development of pacing-induced CHF [8, 13]. For example, Kiuchi and colleagues demonstrated that an early defect in the β-adrenergic transduction system with pacing-induced CHF was reduced adenylate cyclase activity.⁸ In a preliminary study, the change in L-type Ca²⁺ current following exogenous delivery of cyclic AMP remained blunted in pacing-induced CHF myocytes compared to controls [22]. However, direct activation of the L-type Ca²⁺ channel yielded an increase in Ca²⁺ current of similar magnitude in control and pacing-induced CHF groups. Taken together, these results suggest that inherent abnormalities in β-adrenergic receptor mediated phosphorylation of the L-type Ca²⁺ channel must have occurred with the development of pacing-induced CHF. Important intracellular processes for the β-adrenergic mediated modulation of the L-type Ca²⁺ current include activation of guanine nucleotide binding proteins and the phosphorylation state of protein kinase A [23, 26, 34, 35]. In light of the findings from the present study, a future study which directly examines defects in these intermediate steps leading to the phosphorylation of the L-type Ca²⁺ channel during the transition to pacing-induced CHF would be appropriate.

In the present study, the ability of the dihydropyridine, nitrendipine, to bind to the ₁-subunit of the L-type Ca²⁺ channel [15, 30, 37]was utilized to serially measure the relative abundance of the L-type Ca²⁺ channels during the evolution of a CHF process. L-type Ca²⁺ channel densities have been previously examined in clinical and experimental forms of end-stage CHF [15, 30, 38]. In a past study, Colston and colleagues reported that the development of pacing-induced CHF was associated with a 50% reduction in the number of dihydropyridine-binding sites [15]. The present study builds upon these past reports in several important ways. First, the transition to severe pacing-induced CHF was accompanied by reductions in both L-type Ca²⁺ channel density and function. Second, reductions in L-type Ca²⁺ channel density and function were associated with the onset of myocyte contractile dysfunction. Finally, since the present study measured both L-type Ca²⁺ current and abundance, then the relationship between abundance and function of the L-type Ca²⁺ channels could be examined by normalizing the average current recorded from each pig to maximal binding. During the first 2 weeks of rapid pacing, the ratio between L-type Ca²⁺ channel current and abundance was not significantly different from unity. After 3 weeks of pacing and the development of severe CHF, the ratio of L-type Ca²⁺ channel current to abundance fell below unity (0.79, P<0.03). However, it must be recognized that membrane preparations used in the present study were derived from whole myocardial homogenates. While the membrane preparations used in the present study were of equivalent yield and enrichment characteristics, the relative influence of non-myocyte populations on total L-type Ca²⁺ channel binding could not be addressed. Future studies which examine L-type Ca²⁺ channel abundance in isolated myocyte preparations are warranted. Nevertheless, the findings of the present study suggest that with the development of pacing-induced CHF, additional abnormalities in L-type Ca²⁺ channel function occurred over and above an absolute reduction in channel abundance and may have been due to a loss in the Ca²⁺ carrying capacity through residual channels.

Past studies have demonstrated that the ₁-subunit of the L-type Ca²⁺ channel contains the pore and the binding sites for Ca²⁺ channel antagonists [36–38]. Takahashi and colleagues have demonstrated that development of end-stage CHF in humans was associated with reduced mRNA expression of the ₁-subunit of the L-type Ca²⁺ channel [38]. In addition, a past study has demonstrated that the ₁-subunit reverted to the fetal isoform following the development of CHF induced by coronary artery ligation in rats [39]. The ₂- and β-subunits have been demonstrated to play a regulatory role in the determination of L-type Ca²⁺ current magnitude and inactivation kinetics [35, 36]. This laboratory has previously reported that the inactivation time constants for the L-type Ca²⁺ current were altered with the development of severe pacing-induced CHF [18]. Therefore, the absolute reduction in L-type Ca²⁺ channel abundance and function which occurred during the progression of pacing-induced CHF may be due to alterations in protein subunit expression, synthesis, and/or subunit assembly.

Past studies have reported that the development of rapid pacing-induced LV pump dysfunction is associated with abnormalities in myocyte action potential characteristics [18, 40]. In addition to the primary role of the L-type Ca²⁺ current in myocyte excitation–contraction coupling, the influx of Ca²⁺ through these channels determines the duration and morphology of the action potential plateau. This laboratory has recently reported that with the development of pacing-induced CHF, abnormalities in myocyte action potential characteristics, including prolongation of myocyte action potential duration were associated with a reduction in the whole-cell L-type Ca²⁺ current [18]. In a past report by Kääb and colleagues, no differences in steady-state L-type Ca²⁺ current were observed despite prolongation of the action potential duration following chronic pacing in dogs [40]. Nevertheless, this past report did identify that chronic pacing reduced L-type Ca²⁺ current following β-adrenergic receptor stimulation [40]. Direct comparisons between steady-state L-type Ca²⁺ current obtained in this past report and the present study are difficult due to potential differences in the degree of LV dysfunction and methodological considerations. For example, this past study employed the standard, open-patch voltage clamp technique to measure L-type Ca²⁺ currents [40]. However, this method has been previously demonstrated to cause intracellular dialysis and time-dependent, artefactual changes in L-type Ca²⁺ current measurements [26]. In light of the fact that the development of pacing-induced CHF is associated with alterations in intracellular Ca²⁺ homeostatic processes and signal transduction mechanisms [8, 10, 12–16, 18, 19]the nystatin perforated patch clamp technique was used in the present study to minimize artefactual changes in the intracellular milieu [26]. Through this approach, changes in L-type Ca²⁺ current were identified with the progression of pacing-induced CHF. Further, the present study demonstrated that membrane capacitance (an index of cell surface area) was increased with pacing-induced CHF, and this finding is consistent with the significant changes in myocyte geometry which have been reported to occur [8, 13, 18–22, 41]. Interestingly, Kääb et al. did not observe changes in membrane capacitance following chronic ventricular pacing in dogs [40]. In order to determine whether changes in the membrane capacitance would influence absolute L-type Ca²⁺ current during the progression of pacing-induced CHF, the present study examined myocytes of equivalent membrane capacitance. These results confirmed that an absolute reduction in L-type Ca²⁺ current occurred following 2 weeks of rapid pacing and was unlikely due to changes in membrane capacitance. Furthermore, the reduction in the L-type Ca²⁺ current was confirmed by an absolute reduction in L-type Ca²⁺ channel density. While the present study provides evidence that a potential contributory mechanism for the progression of pacing-induced CHF is reduced L-type Ca²⁺ current, there are several important considerations with respect to the L-type Ca²⁺ current measurements. First, the L-type Ca²⁺ currents were measured at room temperature in order to enhance the temporal resolution of the current recordings. Whether similar directional changes in L-type Ca²⁺ current with the progression of pacing-induced CHF would exist under normothermic conditions needs to be established. Second, past studies have demonstrated the existence of species-dependent variability of L-type Ca²⁺ currents in the normal state and with various cardiac pathologies [18, 26–29, 40]. Therefore, extrapolation of the findings from the present study to clinical forms of CHF may be problematic. Finally, it must be recognized that additional defects in myocardial structure and function likely contribute to the initiation and progression of this disease process. For example, a number of ionic changes have been reported with the development of CHF and include reductions in hydrolytic activity of the Na⁺, K⁺-ATPase [10, 19]the transient outward current, and the inward rectifier current [40]. Thus, whether the changes in L-type Ca²⁺ channel abundance and function which occur during the progression of CHF is a manifestation of a more global process remains to be elucidated.

Time for primary review 20 days.

	Acknowledgements

 
This work supported by National Institutes of Health Grant HL45024 (FGS) and a Grant-in-Aid from the American Heart Association. F.G.S. is an Established Investigator of the AHA.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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