Cardiovascular Research

Cardiovascular Research 1998 39(3):589-599; doi:10.1016/S0008-6363(98)00166-7
© 1998 by European Society of Cardiology

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Copyright © 1998, European Society of Cardiology

Altered inotropic response of endothelin-1 in cardiomyocytes from rats with isoproterenol-induced cardiomyopathy

Makoto Suzuki^a, Nobuyuki Ohte^a, Zhong-Min Wang^a, David L. Williams, Jr.^b, William C. Little^a and Che-Ping Cheng^a,*

^aCardiology Section, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1045, USA
^bMerck and Co., Inc., Merck Research Laboratories, West Point, PA 19486, USA

^* Corresponding author. Tel.: +33-6-716-2887; Fax: +33-6-716-9188.

Received 13 January 1998; accepted 26 May 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: The positive inotropic effect of endothelin-1 (ET-1) on normal myocardial contraction may be altered in pathological states. The purpose of this study was to assess the direct effect of ET-1 on cardiomyocyte performance and its cellular mechanism in congestive heart failure (CHF). Methods: We measured the plasma levels of ET-1 and compared the effects of ET-1 (10^–10–10^–8 M) on contractile performance and the [Ca²⁺]_i transient in the myocytes of left ventricles (LV) from 15 age-matched normal adult rats and 15 rats with isoproterenol (ISO)-induced CHF. Results: With CHF, the plasma levels of ET-1 (19.7±6.3 vs. 4.1±0.5 fmol/ml, p<0.05) were markedly elevated. In normal myocytes, superfusion of ET-1 caused significant increases in the systolic amplitude (SA, 8–16%) and the peak velocity of shortening (dL/dt_max, 20–35%; p<0.01) without causing a change in the peak [Ca²⁺]_i transient. In contrast, in myocytes from CHF rats, ET-1 produced significant reductions in SA (9–13%) and in the velocity of relengthening, dR/dt_max (10–14%; p<0.05). The myocytes' dR/dt_max also decreased by 8–10% (p<0.05). These changes were associated with a significant decrease in the peak [Ca²⁺]_i transient (20–23%, p<0.0 1). These responses to ET-1 were abolished by the incubation of myocytes with an ET_A receptor antagonist (BQ123) or a protein kinase C (PKC) inhibitor (H-7 or staurosporine). Conclusion: ISO-induced CHF is associated with elevated plasma ET-1 and an altered cardiomyocyte response to ET-1. After CHF, ET-1 produces a direct depression of cardiomyocyte contractile performance that is associated with a significant decrease in the peak [Ca²⁺]_i transient. These effects are likely to be mediated through ET_A receptors and involve the PKC pathway.

KEYWORDS Rat; Isoproterenol; Congestive heart failure; Endothelin-1; Contraction; Relaxation; Cardiomyocyte; [Ca²⁺]_i transient, Na⁺–H⁺ exchange; Protein kinase C

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Endothelin-1 (ET-1) produced by cardiac myocytes [1]and vascular endothelial cells [2]is present in the plasma at low levels in healthy subjects [1, 2]. In addition to its potent vasoconstrictive effects, ET-1 produces a positive inotropic effect on the intact left ventricular (LV) and isolated myocytes from normal animals [3–5]. Plasma ET-1 concentrations are elevated in congestive heart failure (CHF) [6–9]. It has been demonstrated that high levels of ET-1 alter the vascular response to ET-1. Specifically, the ET-1 induced coronary vasoconstriction is enhanced in both patients and animals with CHF [6, 9]. Recent studies also suggest that the effects of ET-1 on normal myocardial contraction may be altered in pathologic states [10–13]. For example, Sakai and colleagues [14]reported that following coronary artery occlusion in rats, the elevated plasma levels of ET and myocardial accumulation of ET were associated with a decline in LV pressure development. Treatment with ET-1 receptor blocker has been shown to improve cardiac function in CHF and myocardial ischemia [6, 10, 12]. However, the direct effect of ET-1 on myocyte contraction and relaxation has not been previously assessed in CHF [2].

Recently, we observed an altered inotropic response to angiotensin II (ANG II) in conscious dogs with pacing-induced CHF [10]. After CHF, ANG II depressed left ventricular and myocyte contraction and relaxation. The intracellular signaling pathways of ANG II and ET-1 are similar [1, 15, 16]. Thus, the positive inotropic effect of ET-1 on normal myocardium may be altered in a similar fashion in CHF and, thus, contribute to the functional impairment of CHF.

The rat model of isoproterenol (ISO)-induced CHF has been studied by many investigators [17, 18], including those in our laboratory [19]. It has been demonstrated that the pathological changes in ISO-treated rats resemble those of myocardial infarction [18]. Therefore, in the present study, we used this rat model to: (1) determine the effects of ET-1 on myocyte contraction in normal and ISO-induced cardiomyopathic rat hearts and (2) evaluate potential mechanisms of ET-1 inotropic effects in relation to endothelin A (ET_A) receptors, Na⁺–H⁺ exchange, protein kinase C (PKC) activations and [Ca²⁺]_i transients.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Induction and verification of isoproterenol-induced cardiomyopathy
Thirty male Sprague-Dawley rats, weighing 150–200 g (Zivic Miller, Zelienople, NC, USA), were divided into control and CHF groups. The rats in the experimental group received a subcutaneous injection of isoproterenol–HCl (ISO), 85 mg/kg, which was administered on two consecutive days (total of two injections), as previously described [17, 18]. The control groups were injected with equal amounts of sterile saline. The animals were housed and fed under identical conditions for four weeks after the injections. Body weights were obtained before and four weeks after the injections. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publications, No. 85-23, revised 1985).

2.1.1 Hemodynamic measurements
The hemodynamic measurements were performed in ten rats from each group, four weeks after receiving ISO or saline. The rats were then anesthetized with intraperitoneal ketamine HCl (50 mg/kg) and xylazine (10 mg/kg), intubated and ventilated with a positive-pressure respirator (model RSP1002, Kent Scientific, Litchfield, CT, USA) with room air. The right carotid artery was cannulated with a micro-tip pressure transducer (model SPR-249, Millar Instruments, Houston, TX, USA), which was connected to an electrostatic chart recorder (Astro-Med, West Warwick, RI, USA), digitized with an on-line analog-to-digital converter (Data Transition Devices, Boston, MA, USA) at 400 Hz using the Spectrum Program (Wake Forest University School of Medicine, Winston-Salem, NC, USA). Each data acquisition lasted for 10–15 s and spanned several respiratory cycles. The transducer was advanced into the LV for the measurements of LV systolic and diastolic pressures. The time constant of isovolumic LV pressure decay was calculated by the method of Weiss et al. [20]. The maximum time derivative of LV pressure was derived by active analog differentiation of the pressure signal. After LV pressure measurements, a thoracotomy was performed, and a calibrated ultrasonic time-transit flow probe (model 2S, Transonic System, Ithaca, NY, USA) was placed around the ascending aorta to measure aortic flow and to calculate stroke volume.

2.1.2 Plasma endothelin measurements
The plasma endothelin concentrations were measured in five control and five ISO-treated rats, which did not undergo the hemodynamic study. After a light anesthesia, 3–4 ml of blood were obtained from the LV. Immediately, the blood was placed into an EDTA tube on ice and centrifuged at 2500 rpm at 4°C. Plasma was separated and stored at –20°C until assay. A 1-ml volume of 20% acetic acid was added to the 1-ml plasma samples. The acidified samples were vortex-mixed and centrifuged for 15 min at 2600 g. Samples were applied to Si–C₁₈ Sep-pak cartridges (500 mg C₁₈ in a 3-ml syringe) that had been pretreated with 3 ml of metenolone, 3 ml of water and 3 ml of 10% acetic acid. Columns were washed with 3 ml of 10% acetic acid and 6 ml of ethyl acetate. Columns were eluted with 3 ml of 80% methanol–20% 0.05 M ammonium bicarbonate. Eluted samples were dried overnight in a Savant Speed-Vac centrifugal evaporator. Dried samples were assayed for immunoreactive endothelin using an Amersham radioimmunoassay (RIA) kit (RPA 545) [7].

2.2 Cardiomyocyte function evaluation
2.2.1 Myocyte isolation
Ventricular myocytes were enzymatically dissociated using a modification of a previously described technique [21, 22]. After completion of the hemodynamic measurements, the rats that had been anesthetized with intraperitoneal ketamine HCl and xylazine were heparinized (1000 units, i.p.). The heart was quickly excised and immediately placed in iced-cold calcium-free HEPES buffer solution. The heart was perfused via a Langendorff apparatus (No. 508374, Harvard Apparatus, South Natick, MA, USA) with a 100% oxygenated, non-recirculating calcium-free HEPES buffer containing (in mmol/l): NaCl 110.0, KCl 5.4, MgSO₄ 1.2, KH₂PO₄ 1.2, HEPES 10.0, mannitol 45.0 and glucose 15.0. Then the perfusion solution was switched to a 100% oxygenated recirculated HEPES–collagenase solution with 35 µmol/l CaCl₂, 15 mg of collagenase, type II (0.05%, w/v) (144 U/mg, Worthington, Freehold, NJ, USA) and 30 mg of bovine serum albumin (0.1%, w/v) (Fraction V, Sigma, St. Louis, MO, USA) for a total of 30–35 min. Then the flaccid heart was removed from the cannula. The right ventricle, right atrium and left atrium were carefully dissected from the heart, and the left ventricle and remaining myocardium were weighed separately. The LV was minced into 2 mm³ cubes and transferred to a centrifuge tube that contained 10 ml of the HEPES–collagenase solution. It was gently agitated in a water bath for 8–10 min at 36°C. The supernatant was removed and fresh HEPES–collagenase solution was added and incubated for another 8–10 min. This cell isolation procedure produced a mix of LV free wall and septum cells. Then the cells were allowed to settle by gravity. This procedure was repeated three times. The Ca²⁺ concentration of the HEPES buffer was increased in a stepwise fashion (i.e. 250, 500 and 1000 µmol/l). The final pellet was suspended in the modified collagenase-free HEPES buffer (‘the study buffer’), which consisted of (mmol/l): NaCl 137.0, KCl 5.4, MgSO₄ 1.2, KH₂PO₄ 1.2, HEPES 10.0, CaCl₂ 1.2 and glucose 15.0. It was stored at room temperature until it was required for use in the counting of the final yield of viable myocytes and in cell studies. For cell counting, the cells were suspended in HEPES buffer and were dripped into a hemocytometer (Baxter, Hausser Scientific). The cells were counted under a microscope. To obtain the LV weight, the weight of freshly excised rat heart was multiplied by the ratio of the LV weight to the total heart weight, which was obtained immediately after enzymatic digestion of the heart. According to a previous study, this simple calculation permitted an indirect estimate of the non-edematous weight of the LV. In addition, the right kidney weight-to-body weight ratio was used as an index of rat growth [18].

2.2.2 Myocyte morphology
After 2 h of stabilization, the isolated myocytes were placed in superfused culture dishes. The myocytes were imaged with an inverted microscope using a x40 phase-contrast objective. Twenty-five rod-shaped myocytes were measured in each dish.

2.3 Myocyte contractile and [Ca²⁺]_i response to ET-1
2.3.1 Contractile response
In the initial experiments, the effects of ET-1 on the contractile response of myocytes were evaluated. After 1–2 h of stabilization, isolated cardiomyocytes were placed in a cell dish that was continuously superfused with oxygenated study buffer including 1.2 mmol/l Ca²⁺ at 22°C. The myocytes were imaged using a x40 long working distance Hoffman modulation contrast objective attached to an inverted microscope. Myocyte contraction was elicited by field stimulation at a frequency of 0.5 Hz, 1.2 times above the contraction threshold. A video-dimension analyzer with high frame rate (M303 Instrumentations for Physiology and Medicine, San Diego, CA, USA) was used to obtain the trajectory of a moving edge relative to a stationary reference, as well as the distance between two edges. The frequency response of the trajectory of the edge being tracked, as well as that of a varying dimension, was set by the television framing rate (frequency response was linear to 60 Hz, resolution was better than 0.2% of full scale, and linearity was better than 1% of full scale). In the present study, both ends of the cells were tracked. Cardiomyocyte motion and contraction amplitude were digitized with an on-line analog-to-digital converter at 300 Hz. The program ‘SPECTRUM’ (Wake Forest University School of Medicine, Winston-Salem, NC, USA) was used for data analysis. Systolic amplitude or percentage shortening (SA) was determined as the percentage difference between the maximum and minimum cell length of each contraction. The peak velocity of shortening (dL/dt_max) and the peak velocity of relengthening (dR/dt_max) were obtained by differentiating the digitized contractile profiles. Time-to-peak contraction was computed by calculating the time required for the velocity profile to reach zero velocity after the start of contraction. In addition, the velocities of shortening and relengthening were normalized by expressing these parameters as a percentage of the resting cell length [23]. After baseline data collection, cardiomyocytes were superfused with study buffers containing 10^–10–10^–8 M ET-1, and data were obtained again during the 8–10 min ET-1 superfusion period and during the period of wash off of ET-1. To further characterize ISO-induced cardiomyopathy, the response of cardiomyocyte to 10^–8 M ISO was also obtained from both groups.

2.3.2 Intracellular [Ca²⁺]_i response
In another series of experiments, [Ca²⁺]_i and the contraction response of myocytes were measured as previously described [24]. Prior reports [24]have observed that loading cardiac cells with indo-1-AM depresses myocyte contraction. The contractile properties of myocyte could be largely prevented by lighter loading with the probe. Thus, in the current study, a shorter loading period and lower indo-1 concentration were used. After stabilization, isolated myocytes were incubated with 5 µmol/l indo-1-AM (Molecular Probes, Eugene, OR, USA) for 20–30 min. Under these loading conditions, the ratiometric (410/490) fluorescence cell images were homogeneous in both control and ISO-treated cardiomyocytes, indicating that there was no visible intracellular compartmentation of indo-1 [25]. The loaded cells were washed twice with fresh study buffer and left in the dark for 20 min. Then the indo-1-loaded cardiomyocytes were placed in a cell chamber at 22°C and continuously superfused with the study buffer. Approximately 2–3 min were required to change the solution in the cell chamber. A 75-W xenon arc lamp (model LHx75-340, Optical Elements, Sterling, VA, USA) was used to illuminate for epifluorescence, producing 10 µs flashes of 350 nm light at a repetition rate of up to 360 Hz. Paired photomultipliers collected indo-1 emissions by simultaneously measuring spectral windows of 410 and 490 nm, selected by bandpass interference filters. Cells were stimulated at 0.5 Hz for about 20 contractions, and the associated [Ca²⁺]_i was signal-averaged after baseline recording. The ET-1 protocol was repeated as above, and the effect of ET-1 on myocyte contractile response and change of [Ca²⁺]_i were determined.

When cardiomyocytes were loaded with indo-1-AM, compartmentalization of the indicator may have occurred in the mitochondria, thus, the absolute value of [Ca²⁺]_i was not used. In the present study, both 410 and 490 nm wavelengths were expressed by the use of a voltage unit that was fixed at the same gain. One arbitrary unit of fluorescence was calibrated as 10 mv, and the ratio of the emitted fluorescence (410/490) was simultaneously obtained on line through an analog-divider circuit. As used by other investigators [26], this ratio was used to represent the relative changes in peak intracellular [Ca²⁺]_i before and after ET-1 infusion, not the absolute values. In addition, the actual fluorescence ratios were also calibrated as described by O'Neill et al. [27].

2.4 Effects of ET_A receptor, Na⁺–H⁺ exchange, and PKC on ET-1 -induced contractile response
To examine potential mechanisms of ET-1's inotropic effects, myocytes obtained from seven normal control rats and seven ISO-induced CHF rats were divided into three subgroups. One subgroup of myocytes was preincubated with the ET_A-receptor-selective antagonist, BQ123 (1 µmol/l) [28]for 15 min and the second subgroup of myocytes was incubated with an inhibitor of protein kinase C, H-7 (25 µmol/l) or staurosporine (30 nmol/l) [29], for 15 min. To further assess the contribution of Na⁺–H⁺ exchange to ET-1-induced contractile response, the third subgroup of myocytes from both control and CHF rats was incubated with 10^–5 M 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; Sigma), an inhibitor of Na⁺–H⁺ exchanges, for 15 min. After baseline data were collected, ET-1 (10^–8 M) responses were evaluated in all three subgroups. Measurements of myocytes' response were made in 10–20 cells from each animal during each experiment.

2.5 Statistical analysis
All data are presented as mean±SEM. Two-tailed, unpaired Student's t-tests were used to evaluate mean differences in hemodynamic parameters and plasma endothelin concentrations between control and ISO-treated groups. Analysis of the myocyte contractile responses and [Ca²⁺]_i transient data was performed using the average measurements of peak responses obtained from each animal. The control and experimental groups were compared using analysis of variance. If the analysis of variance revealed significance, pairwise tests of individual group mean were compared using Tukey's procedure [30]. The effects of three different concentrations of ET-1 between two different groups of cells were evaluated with ANOVA. Differences were considered to be significant at a value of <0.05.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Characteristics of general features of ISO-induced CHF in adult rats
3.1.1 LV dysfunction and ET levels
As summarized in Table 1, four weeks after ISO injection, LV end-diastolic pressure was doubled. The maximum and minimum time derivative of LV pressure and LV stroke volume were significantly decreased. The rate of LV relaxation slowed, as indicated by a significant increase in the time constant of isovolumic LV pressure decay. The plasma endothelin concentrations in the ISO-treated group were also significantly elevated, compared with the age-matched control group (19.7±6.3 vs. 4.1±0.5 fmol/ml, p<0.05). The animals had clear evidence of CHF (anorexia, edema and pulmonary congestion). There were no significant differences in body weight (428±12 vs. 459±16 g) and heart weight (1.9±0.2 vs. 1.9±0.1 g) between the control and ISO-treated groups. The calculated ratio of LV-to-body weight, (2.4±0.1 vs. 2.6±0.2 g/kg) and the right kidney-to-body weight ratio (3.9±0.1 vs. 4.0±0.2 g/kg) were also not significantly altered. These data are in agreement with those of previous studies [18].

View this table: [in this window] [in a new window]   Table 1 Characterization of isoproterenol-induced LV and cardiomyocyte dysfunction in adult rats

 
3.1.2 Cardiomyocyte dysfunction
ISO-treated hearts were more easily digested by collagenase than control hearts, and the isolation time was shorter than that in control hearts. The first harvest of the rod-shaped cells per heart in the ISO-treated rat hearts was lower than in the control group. Although the final yield of viable myocytes in the ISO-treated rats tended to be lower, the difference between the two groups did not reach statistical significance [81±8% vs. 79±8%, p=not significant (NS)]. These findings are consistent with previous observations and our recent study [10]. At room temperature (22°C), the isolated myocytes maintained rod-shape morphology for more than 12 h. Compared with control, the resting myocyte length (131.7±1.5 vs. 127.6±1.7 µm) and width (27.6±0.5 vs 25.7±0.6 µm) of the ISO-treated myocytes were not significantly altered. However, as shown in Table 1Fig. 1A, cardiomyocyte contractile function was markedly depressed. In the ISO-treated cardiomyocytes, the baseline SA, dL/dt_max and dR/dt_max were significantly depressed. The normalized peak velocity of shortening (139±20 vs. 90±24%) and relengthening (89±36 vs. 56±19%) remained significantly lower in the ISO-treated myocytes than in the controls. Time to peak SA and half-relengthening time were also prolonged. As presented in Fig. 1B, the depressed myocyte contractile performance in the ISO-treated group was associated with a significant decrease in peak systolic [Ca²⁺]_i (1.5±0.1 vs. 1.8±1.0). In addition, after ISO-treatment, myocytes had a depressed response to β-adrenergic stimulation. The exposure to 10^–8 M ISO in the normal myocytes resulted in an increase in contractile performance, with significant increases in SA (25.2±5.5%), dL/dt_max (73.7±12.1%) and dR/dt_max (21.9±8.8%). In contrast, in ISO-treated myocytes, these responses to ISO were significantly attenuated and the ISO-induced increases in SA (11.9±3.3%), dL/dt_max (33.1±6.8%) and dR/dt_max (1 2.6±7.5%) were significantly reduced.

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Representative superimposed traces of analog recording of contractile response (A) and averaged [Ca2+]i transients response (B) in the isolated myocytes obtained from one normal control rat and one ISO-treated rat. Myocytes were superfused with HBS solution at 22°C and field-stimulated at 0.5 Hz. (A) Compared with normal myocyte, the percent of shortening, the peak velocity of shortening and relengthening were all markedly reduced in the ISO-treated myocyte. (B) In the ISO-treated myocyte, during field stimulation, resting [Ca2+]i was elevated, peak systolic [Ca2+]i was markedly decreased.

 
3.2 Contractile and [Ca²⁺]_i transient response of cardiomyocytes to endothelin-1
The peak responses of ET-1 are displayed in such a way in Fig. 2 as to show that superfusion of ET-1 (10^–10–10^–8 M) in the normal myocytes caused 7.8±2.8, 8.7±2.8 and 16.0±3.3% increases in SA, respectively, and 20.5±4.7, 17.7±6.4 and 34.4±7.9% increases in dL/dt_max. The velocity of relengthening, dR/dt_max was not significantly changed with 10^–10 and 10^–8 M ET-1 (7.3±0.9 and 9.7±1.2%, respectively), but increased with 10^–9 M ET-1 (11.6±1.1%). In contrast, in myocytes from ISO-treated rats, ET-1 (10^–10–10^–8 M) produced about 8.9±1.9, 11.4±2.8 and 12.9±3.2% decreases in SA, respectively (p<0.05). ET-1 also caused 10.9±2.3, 12.5±3.9 and 14.4±4.2% (p<0.05) decreases in dL/dt_max, and 8.2±1.9, 7.4±2.9 and 9.8±3.6% (p<0.05) decreases in dR/dt_max.

View larger version (33K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 (A) An example of contractile response to ET-1 in the myocytes obtained from one normal rat and one ISO-treated rat. In the normal myocyte, ET-1 (10–8 M) increased systolic amplitude and the peak velocity of shortening. In contrast, in the ISO-treated myocyte, ET-1 caused decreases in the systolic amplitude, and in the peak velocity of shortening and relengthening. (B) Group mean data of contractile response to ET-1 (10–8 M) of normal and ISO-treated cardiomyocytes from ten control and ten experimental animals.

 
The viable myocyte yield from the indo-1 loading was comparable to that from conventional myocyte preparations without the dye. Compared with unloaded control myocytes, in both control and ISO-treated groups, the SA (control: 16.6±1.6 vs. 15.9±2.1; CHF: 13.0±1.7 vs. 12.2±2.1%) and time to peak contraction (control: 201.7±3.8 vs. 209.8±4.2; CHF: 253.1±3.9 vs. 258.6±4.8 ms) were not significantly altered in the indo-1-loaded cells. As shown in Fig. 3, in the normal myocytes, ET-1 caused no significant changes in the systolic amplitude of [Ca²⁺]_i transient. However, in the CHF myocytes, ET-1 (10^–10–10^–8 M) resulted in significant reductions in the amplitude of the systolic [Ca²⁺]_i transients (22.8±4.9, 20.1±5.0 and 21.6±3.9%, respectively, p<0.05). These inotropic effects of ET-1 occurred in 4–6 min and reached the maximum contractile response after 7–10 min of superfusion of ET-1. These effects were nearly completely returned to control levels after washout of the ET-1.

View larger version (33K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 (A) An example of averaged calcium transients response to ET-1 in normal and ISO-treated cardiomyocytes. In response to ET-1, the systolic peak calcium transients was relatively unchanged in the normal cardiomyocyte, but was markedly decreased in the ISO-treated cardiomyocyte. (B) Group mean data of calcium transient response to ET-1 of normal and ISO-treated cardiomyocytes from ten animals in each group. After ET-1 superfusion, the peak systolic of calcium transients in normal cardiomyocytes was relatively unchanged, but it was significantly decreased in ISO-treated myocytes.

 
3.3 Effect of ET_A receptor, Na⁺–H⁺ exchange and PKC on the ET-1-induced contractile response
To investigate the potential mechanisms of ET-1-induced inotropic effects in relation to the ET_A receptor, Na⁺–H⁺ exchange and PKC activation, in the third serial study, cardiomyocytes from both normal and CHF groups were randomly preincubated with an ET_A receptor blocker, BQ123, a Na⁺–H⁺ exchange inhibitor, EIPA, and a PKC inhibitor, H-7 or staurosporine.

3.3.1 Effect of ET_A receptor
As presented in Fig. 4, compared with unincubated cells, pretreatment with BQ123 resulted in no significant changes in SA and systolic peak [Ca²⁺]_i transient. However, pretreatment with BQ123 resulted in almost complete inhibition of the ET-1-induced contractile and [Ca²⁺]_i transient responses.

View larger version (51K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Group mean data of the effects of BQ123 (A) and H-7 (B) on ET-1-induced inotropic and [Ca2+] transient responses in the normal and ISO-treated cardiomyocytes from seven control and seven experimental animals. Pretreatment with BQ123 and H-7 resulted in no significant changes in baseline contractile function and peak systolic [Ca2+]i transient, but prevented ET-1-induced effects in both normal control and ISO-treated groups.

 
3.3.2 Effect of Na⁺–H⁺ exchange
Compared with unincubated cells, pretreatment with EIPA had no significant effect on baseline values of SA and systolic peak [Ca²⁺]_i transient in both normal and CHF myocytes. In the normal myocytes pretreated with EIPA, ET-1 (10^–8 M) failed to produce significant changes in SA (16.3±1.9 vs. 16.1±1.6%), dL/dt_max (180.3±14.4 vs. 176.2±17.6 µm/s) and dR/dt_max (107.9±24 vs. 111.9±21 µm/s). In contrast, in the CHF myocytes that were pretreated with EIPA, the ET-1-induced reductions of SA (13.9±2.1 vs. 11.6±2.6%), dL/dt_max (123.8±4.5 vs. 104.5±6.4 µm/s) and dR/dt_max (73.8±3.3 vs. 67.8±4.9 µm/s) (p<0.05) persisted.

3.3.3 Effect of PKC inhibitor
As shown in Fig. 4, cardiomyocytes that had been preincubated with H-7 prevented any significant changes in the contraction and [Ca²⁺]_i transient in response to 10^–8 M ET-1. A similar observation was also made in the myocytes of both groups that had been pretreated with staurosporine. In the normal cardiomyocytes, after pretreatment with staurosporine, the SA was 17.6±1.7%. With the addition of ET-1 (10^–8 M), the SA remained relatively unchanged (17.4±1.3%, p=NS). The calcium transient systolic amplitude was also not altered with ET-1 (1.7±0.8 vs. 1.8±1.1, p=NS). In the CHF myocytes, after pretreatment with staurosporine, ET-1 also failed to produce changes in SA (13.8±1.6 vs. 13.5±2.7%, p=NS) and systolic peak [Ca²⁺]_i transient (1.5±1.1 vs. 1.5±1.6, p=NS).

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Our study demonstrates that ET-1 evokes a positive inotropic response in normal myocytes without causing a change in the systolic amplitude of the [Ca²⁺]_i transient; however, in myocytes from rats with ISO-induced CHF, ET-1 produces a direct depression in contraction and relaxation and reduces the peak systolic [Ca²⁺]_i transient. These effects are mediated through ET_A receptors and involve the PKC pathway.

4.1 Isoproterenol-induced CHF model
The rat model of ISO-induced CHF has been studied by many investigators [17–19]. High doses of ISO produce a time- and dose-dependent impairment of cardiac function that results in CHF. Teerlink et al. [18]reported that the pathological changes in ISO-treated rats resemble those of myocardial infarction. Consistent with previous studies, we found that four weeks after two daily injections of 85 mg/kg isoproterenol, all of the ISO-treated rats had evidence of CHF (anorexia, edema and pulmonary congestion), with LV systolic and diastolic dysfunction, as indicated by significant increases in LVP_ED, min LVP and , and decreases in SV, ±dP/dt_max and stroke work (Table 1). The plasma ET levels were also significantly elevated with ISO treatment. However, there was no LV hypertrophy. Previously, Teerlink et al. [18]reported that, by six weeks after the injection of 85 mg of ISO, the increased LV filling pressure was associated with myocardial hypertrophy and ventricular dilation. The lack of LV hypertrophy in our animals could be due to a shorter duration of observation after ISO administration.

Compared with normal myocytes, the myocytes from the ISO-treated rats showed defects in the contractile performance and [Ca²⁺]_i homeostasis, as indicated by the significantly reduced systolic amplitude (SA) and velocity of contraction (dL/dt_max), velocity of relengthening (dR/dt_max) and the peak systolic [Ca²⁺]_i transient (Table 1Fig. 1). In addition, the myocyte contractile response to β-adrenergic stimulation was also significantly blunted. The alterations in myocyte function were similar to those observed in myocytes from patients with CHF and some other animal models of CHF [10, 31].

4.2 Inotropic effect and basic cellular mechanism of ET-1 in normal and ISO-induced CHF
To obviate the confounding influence of extracellular factors, we studied freshly isolated myocytes from the LV of normal and ISO-treated rat hearts. In the normal adult myocyte, ET-1 (10^–10–10^–8 M) exerted a positive inotropic effect, as indicated by the increases in SA and dL/dt_max (Fig. 2). This is consistent with previous studies of ET-1 using papillary muscles [5], cardiomyocytes [3, 4, 16]and conscious animals [32]. In contrast, the myocytes obtained from ISO-treated rats had a different response to ET-1. In the ISO-treated myocytes, ET-1 decreased SA, dL/dt_max and dR/dt_max. These findings are in agreement with a preliminary report by Thomas et al. [11], who reported a similar dose-dependent negative inotropic effect with ET-1 (1–400 pmol/l) in isolated myocytes from pigs with pacing-induced CHF. These observations are also compatible with several studies that showed that ET-1 receptor blockade improves LV and myocardial function in CHF [8, 12, 33]. Our results differ from the work of Li and Rouleau [34]and Sakai et al. [14]. Li and Rouleau [34], using isolated papillary muscles from normal dogs and from dogs with CHF that was induced by pacing, found that ET-1 caused a positive inotropic response [34]. Similarly, Sakai et al. [14], in an anesthetized left coronary artery ligated rat model of CHF, reported that blocking the ET_A receptor with BQ123 induced a negative inotropic action [14]. These inconsistent results may have resulted from the influence of ET-1 or ET-1 receptor blocker-produced changes in loading conditions on conventional measurement of LV performance, variable effects of anesthesia and different levels of CHF [8].

The amount of ET-1 we used was similar to the amount used in previous studies [5, 15]. However, this produces ET-1 levels that are well above the plasma levels in the ISO-treated rats. These experiments may still be relevant because the tissue levels of ET-1 are much higher than the plasma ET-1 concentration [14, 35]. For example, Loffler et al. [35]found that the ET-1 concentration in ventricles was roughly four orders of magnitude higher than the plasma concentration in normal rabbits. Furthermore, in CHF, the myocardial ET system is up-regulated, producing much higher cardiac ET-1 levels than in normals [6, 7, 14].

Kramer et al. [4]found that the positive inotropic effect of ET-1 in normal hearts is due to stimulation of the sarcolemmal Na⁺–H⁺ exchanger, mediated by the protein kinase C (PKC) pathway. This leads to intracellular alkalosis and sensitization of the cardiac myofilaments to intracellular Ca²⁺ [4]. Our findings of the lack of a change in the peak amplitude of the [C²⁺]_i transient accompanying the positive inotropic effect of ET-1 in the normal myocytes are consistent with this mechanism. We further observed that pretreatment of normal myocytes with EIPA prevented the ET-1-induced positive inotropic response. It appears that, in adult normal rat myocytes, ET-1 caused a positive inotropic effect due to stimulation of the Na⁺–H⁺ exchange.

After ISO-induced CHF, we found that the negative inotropic effect of ET-1 was associated with a decrease in the peak systolic Ca²⁺ transient that was not present in normal myocytes and was not altered by pretreatment with an inhibitor of Na⁺–H⁺ exchange. However, an inhibitor of PKC blocked the negative inotropic effect. These observations suggest that the response to ET-1 activation of the PKC pathway is altered in CHF myocytes, producing less Ca²⁺ release that results in less systolic force generation [36–39]. In addition, PKC activation may reduce myofilament Ca²⁺ sensitivity [13, 40]and contribute to the negative inotropic response to ET-1 in the ISO-induced CHF myocytes. In a recent study, Ito et al. [13]reported a similar finding of an altered inotropic response to ET-1 in cardiac hypertrophy. They found that ET-1 failed to promote a positive inotropic response in hypertrophied myocytes due to an impaired ET-1 stimulation of Na⁺–H⁺ exchange. There is a defect in the coupling of PKC activation with enhanced Na⁺–H⁺ exchange in hypertrophied myocytes [13]. PKC has been shown to phosphorylate both the inhibitory subunit of troponin I (TnI) and the tropomyosin-binding subunit, troponin T (TnT) [38, 41, 42]. It has been demonstrated that TnI phosphorylation reduces myofibrillar Ca²⁺ sensitivity and plays an important role in the pH regulation of Ca²⁺-force relation in cardiac muscle. It is possible that, in CHF myocytes, the phosphorylation of TnI by PKC outweighs the ET-1-induced alkalinizing effect, resulting in a net decrease in the sensitivity of the myofilaments, thus contributing to the altered inotropic response to ET-1 in CHF myocytes.

Previously, activation of PKC has been shown through phosphorylation-dependent mechanisms to alter Ca²⁺ transporters [36], Ca²⁺ homeostasis and intracellular Ca²⁺ [37], and to modulate myofibrillar Ca²⁺ sensitivity [13, 38]. Recent studies suggest that PKC activity and expression may be altered in CHF. An enhanced PKC activity has been demonstrated in myocardial ischemia and in pressure-overload cardiac hypertrophy [43, 44], and an altered PKC activity and expression of a Ca²⁺-dependent PKC isoform have also been reported in rabbits with CHF [45]. Thus, we speculate that the ET-1-induced PKC activity may also be altered in the ISO-treated rats, which may lead to alterations of PKC-induced phosphorylation and, thus, further depress the mobilization and reuptake of [Ca²⁺]_i and myofibrillar Ca²⁺ sensitivity [13, 36–38, 46]. Our observation of an involvement of PKC in mediating the ET-1-induced inotropic response of cardiomyocytes is consistent with a number of past reports [4, 13, 47].

Although not a universal finding [14], several in vitro studies demonstrated a down-regulation of the ET-1 receptor in the failing heart. There is also a desensitization of the ET-1 transmembrane signaling pathway in the coronary arteries in CHF [35]. Specifically, Loffler et al. [35]found, in rabbits with CHF, that when the plasma ET was elevated, the density of ET-1 receptors in the heart and the kidney was decreased [35]. However, desensitization of ET-1 receptors cannot account for the altered cardiomyocyte response to ET-1 in the ISO-treated rats, since the nature of the response of the ISO-treated myocytes to ET-1 was altered. Blocking the ET_A receptors with BQ123 almost completely prevented the positive inotropic effects of ET-1 in normal myocytes and the negative effect in ISO-treated myocytes.

Kohmoto et al. [46]found that immature myocytes have the same negative inotropic response to ET-1 that we observed in the ISO-induced CHF myocytes. This similarity is consistent with the recapitulation of the neonatal phenotype in heart failure [39]. It has been reported that many regulatory proteins, such as TnT, as well as PKC, undergo maturationally and regionally regulated expression in the heart, and there is a recapitulation of the neonatal phenotype in some disease states [39].

Several methodologic issues should be considered in interpreting our data. First, we used enzymatic dissociation to isolate the myocytes from rat hearts. Since not all cells recover after enzymatic dissociation, the potential exists that the sampling was biased towards those cells that survived. This bias could be further complicated by the possibility that a different sub-population of cells survived from the ISO-treated heart tissue. However, previous studies have reported that the individual myocytes isolated by this technique retain morphological and contractile performances that are similar to those observed in the intact muscle [22]. Consistent with previous observations [21, 22], we have obtained a high yield of viable myocytes by this preparation of the LV from both normal and ISO-treated rat hearts. Furthermore, evidence of ISO-induced changes is clearly demonstrated by the alteration in the morphology, contraction and relengthening, [Ca²⁺]_i, and depressed responses to β-adrenergic stimulation of myocytes isolated from ISO-treated rat hearts. Thus, our observation of an altered response to ET-1 in ISO-treated myocytes is unlikely to be due to sampling bias or to artifacts introduced by the enzymatic isolation process.

Second, the compartmentalization of dye in organelles is a major problem in the estimation of cytoplasmic [Ca²⁺]_i. To minimize the accumulation of dye in organelles, we used indo-1 and relatively short loading periods and performed experiments at room temperature. In addition, the images for control and ISO-treated myocytes were homogeneous, indicating that there was no visible intracellular compartmentation of indo-1. However, it is possible that there was some accumulation of dye in organelles, which could contribute to an underestimation of [Ca²⁺]_i in this study.

In conclusion, we found that ISO-induced CHF is associated with an altered myocyte response to ET-1. In contrast to its positive inotropic and myofilament-sensitizing effect in normal myocytes, ET-1 produces a direct depression in CHF myocytes' contractile performance, with a reduced [Ca²⁺]_i. Our results suggest that these effects are mediated through activation of the ET_A receptor, modulated by activation of the PKC pathway, and may contribute to functional impairment in CHF.

Time for primary review 23 days.

	Acknowledgements

 
This study was supported in part by grants from the National Institutes of Health (HL45258 and HL53541), the American Heart Association (94006140 and 9640489N), and the Alcoholic Beverage Medical Research Foundation. Dr. Cheng is an Established Investigator of the American Heart Association. We gratefully acknowledge the computer programming (SPECTRUM) of Ping Tan, the technical assistance of Drs. T. Ukai, K. Onishi, and Z.S. Zhang, and Mack Williams, and the secretarial assistance of Carol S. Corum.

	References

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