Cardiovascular Research

Cardiovascular Research 1999 44(2):398-406; doi:10.1016/S0008-6363(99)00205-9
© 1999 by European Society of Cardiology

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Copyright © 1999, European Society of Cardiology

Role of Ca²⁺ availability to myofilaments and their sensitivity to Ca²⁺ in myocyte contractile dysfunction in heart failure

Shintaro Kinugawa, Hiroyuki Tsutsui^*, Shinji Satoh, Masaru Takahashi, Tomomi Ide, Keiko Igarashi-Saito, Ken-ichi Arimura, Kensuke Egashira and Akira Takeshita

Research Institute of Angiocardiology and Cardiovascular Clinic, Kyushu University School of Medicine, Fukuoka 812-8582, Japan

^* Corresponding author. Tel.: +81-92-642-5360; fax: +81-92-642-5374 prehiro{at}cardiol.med.kyushu-u.ac.jp

Received 8 March 1999; accepted 7 June 1999

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Contractile function is depressed at the isolated myocyte level in heart failure (HF), which could result from the decreased availability of intracellular calcium ([Ca²⁺]i) to the myofibrils and/or the depressed sensitivity of myofilaments to [Ca²⁺]i. However, the cellular basis of contractile dysfunction remains unestablished. Methods: We isolated left ventricular myocytes from dogs with rapid pacing-induced HF. Cell shortening and [Ca²⁺]i transients were measured by indo-1 fluorescence and the myofilament Ca²⁺ sensitivity was analyzed by the shortening–[Ca²⁺]i relation in intact myocytes as well as by the pCa–tension relation in skinned cells. Results: Peak cell shortening magnitude was depressed in HF, associated with a parallel decrease of [Ca²⁺]i transient amplitude. There was a significant positive correlation between these two variables (r=0.71, P<0.01). In contrast, myofibrillar sensitivity to Ca²⁺, determined by both intact and skinned myocytes, was comparable between control and HF. Further, there was no significant difference in Ca²⁺ sensitivity between control and HF even at shorter (1.8 µm) or longer (2.2 µm) sarcomere length. Conclusions: Using both intact and skinned cellular preparations, a potential defect in myocyte contractile function in HF was a reduction in Ca²⁺ availability to the myofilaments, rather than the inherent defects in myofilament sensitivity to Ca²⁺.

KEYWORDS Calcium (cellular); Cell culture/isolation; Contractile function; Heart failure; Myocytes

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Contractile function is impaired at the isolated cardiac muscle cell (myocyte) level in failing hearts [1], which suggests that the intrinsic abnormalities within myocytes are responsible for the pathogenesis of heart failure (HF). Recent studies in the experimental animal model of cardiac hypertrophy and failure demonstrated that the diminished myocyte contraction was associated with small and prolonged intracellular Ca²⁺ ([Ca²⁺]i) transients [2–4]. Similar changes in [Ca²⁺]i transients have been also demonstrated in myocytes isolated from end-stage human HF [5]. These findings would support the hypothesis that the decrease in systolic [Ca²⁺]i transient is responsible for the contractile dysfunction in HF. However, it has not been determined whether the changes in [Ca²⁺]i homeostasis are sufficient to explain abnormalities in myocyte contractility.

In addition to the alterations in [Ca²⁺]i transients, the decrease in the sensitivity of myofilament to [Ca²⁺]i could be responsible for the contractile dysfunction in HF. However, to date, measurements of myofibrillar Ca²⁺ sensitivity using papillary muscles or ventricular myocytes from failing hearts have yielded conflicting results, showing to be unchanged [2,6,7], decreased [3,8,9], and even increased [10,11]. The reasons for these discrepant results could be related to the differences in the types of preparations studied; i.e. multicellular tissue vs. single cell preparations. We consider that the isolated myocyte preparations are suitable for the assessment of [Ca²⁺]i compared to papillary muscle or whole heart since the fluorescent signal of Ca²⁺ measured on the epicardial surface of the multicellular tissue might not accurately reflect the [Ca²⁺]i transients in myocytes. In addition, the type of preparations, intact vs. skinned, is also responsible for these differences. We thus consider that it is critically important to assess myofibrillar Ca²⁺ sensitivity in intact as well as skinned myocyte preparations and to compare both results obtained from the same heart. However, to date, no studies have assessed myofibrillar Ca²⁺ sensitivity concurrently in both intact and skinned preparations.

The purpose of the present study was to examine whether there were significant differences in [Ca²⁺]i transients and contractions between normal and failing myocytes bathed in solutions with physiological Ca²⁺ concentration and the changes in the magnitude and duration of [Ca²⁺]i transient could be sufficient to explain the contractile defects of failing myocytes. Further, we examined whether the decrease of myofibrillar Ca²⁺ sensitivity might be also involved for the myocyte contractile dysfunction as an additional factor in both intact and skinned preparations.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Preparation of animal models
HF was induced in seven adult mongrel dogs (16 to 26 kg body weight) by rapid ventricular pacing (HF dogs) according to the methods described previously [12,13]. Briefly, under general anesthesia, a bipolar pacing lead (1236T; Pace Setter) was introduced into the external jugular vein and placed in the right ventricle under fluoroscopic guidance. After recovery from the surgery, rapid ventricular pacing at 240 beats per min was begun and maintained continuously for 4 weeks. Control dogs were treated in an identical manner as HF dogs, in which a pacing lead was inserted without pacing (n=10).

2.2 In vivo LV function
On the day of the study, ventricular pacing was stopped and, under general anesthesia, the animals were intubated and ventilated with a respirator on a heating pad to maintain the body temperature at 37°C. LV contractile performance was assessed by LV pressure and echocardiograms as described previously in detail [12,13]. All procedures and animal care were reviewed and approved by the Committee on Ethics of Animal Experiments, Faculty of Medicine, Kyushu University and conducted according to the Guidelines for Animal Experiments of the Faculty of Medicine, Kyushu University and Law (No. 150) and Notification (No. 6) of the Japanese Government.

2.3 Simultaneous measurement of shortening and [Ca²⁺]_i transient in intact myocytes
Cardiac myocytes were isolated from the LV free wall by perfusing collagenase as described previously [12,13]. [Ca²⁺]_iwas measured by using the fluorescent Ca²⁺ indicator dye indo 1 under electrical field stimulation (0.25 Hz) at 35°C according to the methods described previously [14]. The indo-1 fluorescence ratio was calibrated using an in situ method and expressed as the absolute values of [Ca²⁺]i from the following equation; [Ca²⁺]i=K_d·β·[(R–R_min)/(R_max–R)], where R_min and R_max are the fluorescence ratios at zero and saturating [Ca²⁺]i, respectively; K_d is the dissociation constant (250 nmol/l), and β is the fluorescence ratio of the 485-nm signal in low Ca²⁺ to that in high Ca²⁺. Cell autofluorescence was determined in cells in a nominally Ca²⁺-free solution containing 10 mmol/l Mn²⁺ and 2 µmol/l ionomycin and was subtracted before calculating other constants.

Myocytes were simultaneously illuminated with red light (wavelength above 620 nm) through the normal bright-field illumination optics of the microscope. The cell image was projected onto a photodiode array of the edge detector (C6294-01; Hamamatsu Photonics) and the cell length was monitored simultaneously with indo-1 fluorescence under electrical field stimulation (0.25 Hz) at 35°C. The contractile state of individual myocytes was assessed by measuring the magnitude of cell shortening, which was defined as the diastolic cell length (DCL), the longest cell length measured during the diastole, minus the cell length at peak shortening during each beat divided by DCL for that beat and expressed as a percentage of DCL [15].

We measured maximal Ca²⁺-activated shortening by tetanizing ryanodine-treated intact cells according to the methods described by Yue et al. [16] with some modifications [17] and thereby deduced myofilament Ca²⁺ sensitivity. Using indo-1-loaded myocytes, we assessed simultaneously steady-state cell length and steady-state [Ca²⁺]i. Cell tetani were induced by stimulating intact myocytes at 10 Hz for 4–6 s in the presence of ryanodine (10 µmol/l). Steady-state measurements were made during the first 4–6 s of the tetani. Tetani were reproducible when they were separated by an interval of at least 3 min during which cells were stimulated at 0.25 Hz. Tetani were elicited at varying [Ca²⁺]o from 0.625 to 20 mmol/l. Phosphate was removed from the bathing medium during these measurements to avoid calcium precipitation. The cell shortening versus [Ca²⁺]i curves were fit to a modified Hill equation [18]; log [S(1–S)]=n(log [Ca²⁺]+k), where S is the magnitude of cell shortening as a fraction of maximal Ca²⁺-activated shortening, n is the Hill coefficient, and k is the x intercept of the fitted line, which corresponds to the Ca²⁺ concentration at which shortening is half maximal (pCa₅₀).

2.4 Measurement of tension–pCa relation in skinned myocytes
To obtain the skinned myocyte preparations, the cellular membrane was removed by incubating the intact myocytes with 25 µg/ml of β-escin for 10 min at 4°C in the relaxing solution containing (in mmol/l) K-methanesulfonate (K-MS) 110, Mg(MS)₂ 5, Na₂ATP 5, EGTA 4, creatine phosphate 4, and PIPES 20 according to the methods described previously with some modifications [19]. We have recently demonstrated that the adrenergic receptor function is preserved normal in these cellular preparations [19]. Ionic strength was adjusted to 200 mmol/l with KCl and pH was adjusted to 7.1. The skinned myocytes with clear uniform sarcomere pattern were kept in the chamber mounted on the mechanical stage of an inverted microscope on a vibration-free table. The stainless steel needles (tip diameter, 1 to 2 µm) were gently stuck to both ends of the myocyte in Ca²⁺-free relaxing solution. One end of the cell was impaled and fixed to the rubber bottom of the chamber and the other end was attached to a microforce transducer (model AE801, Aksjeselskapet). The myocyte image was observed on a TV monitor through a CCD camera (model XC-77; Sony) and both sarcomere and myocyte length were measured by using a computer-assisted image analysis (model SVS 3000; Showa Electric) during myocyte relaxation and activation. Myocyte cross-sectional area was calculated using cell width on the assumption of an elliptical cross-section. The temperature of the chamber was maintained at 25°C by a thermostated heating stage (model DC-MP10DM; Kitazato).

After attaching the myocyte to the experimental apparatus, sarcomere length was initially set at 2.0 µm and monitored continuously during the activation. Myocytes were treated first with ryanodine (30 µmol/l) and caffeine (30 mmol/l) in pCa 6 solution (pCa: –log Ca²⁺ concentration) for 4 min and then washed in the relaxing solution to inhibit Ca²⁺ release function of the sarcoplasmic reticulum (SR). Then, to obtain tension–pCa relations, the skinned myocytes were activated by increasing the Ca²⁺ concentrations of the bathing solutions cumulatively from pCa 9.0 (relaxing solution) to 5.0 (maximally activating solution). The cells with sarcomere length, which varied by more than 10% of initial value were discarded. The force at maximum activation was compared at the beginning and end of pCa changes and, when the final force was less than 90% of initial force, the sequence was not used in the analysis. The Ca²⁺ sensitivity of tension and degree of cooperative activation, as judged by the steepness of the tension–pCa relation, were determined by least-square regression using the Hill transformation to linearize the sigmoidal tension–pCa relations [18]; log [P(1–P)]=n(log [Ca²⁺]+k), where P is active tension as a fraction of maximal Ca²⁺-activated tension, n is the Hill coefficient, and k is the x intercept of the fitted line, which corresponds to the Ca²⁺ concentration at which tension is half maximal (pCa₅₀).

To confirm that the above methods could detect the alterations in myofibrillar Ca²⁺ sensitivity if they were present, the effects on these measurements of pharmacological intervention known to produce primary alterations in myofibrillar Ca²⁺ sensitivity were examined in normal myocytes; EMD57033 to increase sensitivity [20] and isoproterenol or butanedione monoxime (BDM) to decrease it [21].

2.5 Statistical analysis
All data were presented as the means±SE. An unpaired Student's t-test was used to compare values between control and HF. Changes in cell shortening and [Ca²⁺]i transient at different extracellular Ca²⁺ concentrations and pCa-tension relations were compared between control and HF by one way ANOVA for repeated measures. For linear regression data, the correlation coefficient was calculated using a least-squares fit analysis. Differences were considered statistically significant at P<0.05.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 LV contractile function
Body weight (19±3 vs. 23±3 kg, P=NS) and LV weight (85±11 vs. 85±14 g, P=NS) did not differ significantly. Chronic rapid pacing caused an approximately 50% increase in LV end-diastolic dimension (35±3 vs. 50±1 mm, P<0.01), a 107% increase in LV end-systolic dimension (21±2 vs. 44±1 mm, P<0.01), and a 60% decrease in LV ejection fraction (73±3 vs. 29±2%, P<0.01) by echocardiography. For the HF dogs, LV peak +dP/dt was significantly depressed (2400±181 vs. 1480±134 mmHg/s, P<0.01) and LV end-diastolic pressure (5±1 vs. 32±8 mmHg, P<0.01) was significantly increased compared to control values.

3.2 Cell shortening and [Ca²⁺]i transient in intact myocytes
The average yield of rod-shaped myocytes was the same between control and HF (70±3 vs. 73±4%, P=NS). The ratio of rods responding to electrical field stimulation to all rods was also comparable between two groups (90±3 vs. 88±4%, P=NS). We confirmed no significant differences (P=NS by unpaired t-test) between two groups of myocytes (Table 1) in autofluorescence level, calibration constants and the relative proportion of dye in the organelles (namely mitochondria) by the Mn²⁺ quench technique [22], indicating that the comparisons of calculated [Ca²⁺]i concentration values between control and HF are valid. In the presence of indo-1, cell shortening was slightly decreased to a similar extent in control and HF myocytes (9.6±0.9 vs. 10.1±0.9% decrease from pre-indo-1 levels, P=NS), indicating that Ca²⁺-binding effects of indo-1 were comparable.

View this table: [in this window] [in a new window]   Table 1 Comparison of properties of indo-1 in control and HFa

 
Representative simultaneous tracings of cell shortening and [Ca²⁺]i transient in the externally unloaded intact myocytes are shown in Fig. 1. Table 2 represents a summary of the data for the characteristics of myocyte shortening and [Ca²⁺]i transient dynamics from these two groups. In comparison with controls, HF myocytes demonstrated that the cell shortening magnitude was depressed by 62% (P<0.01), which was associated with a 36% reduction in the amplitude of [Ca²⁺]i transient (P<0.01), and the 50 and 90% relaxation time were increased by 236 and 180% (P<0.01), respectively, which was associated with a prolonged decline (130 and 56%, respectively, P<0.05) of the [Ca²⁺]i transient. There was no significant difference in the resting levels of [Ca²⁺]i between the two groups.

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Representative simultaneous recording of cell shortening (above) and [Ca2+]i transient (below) in intact myocytes isolated from control (left) and HF (right) dogs. Note that cell shortening was reduced in HF, which was associated with the reduction in [Ca2+]i transient amplitude. Duration of cell shortening and [Ca2+]i transient was prolonged in HF myocyte.

 

View this table: [in this window] [in a new window]   Table 2 Characteristics of myocyte shortening and [Ca2+]i transient dynamicsa

 
Fig. 2A shows the relationship between the magnitude of cell shortening and the amplitude of the [Ca²⁺]i transient. Each data point represented for each animal, which consisted of the average data for four to six myocytes. There was a significant positive correlation between two parameters in control and HF (r=0.71, n=17 hearts, P<0.01). Further, there was a significant positive correlation between the duration of cell shortening and that of [Ca²⁺]i transient (Fig. 2B and C). These results suggest that the abnormalities in [Ca²⁺]i transient amplitude and duration were in parallel to the reduced and prolonged contraction of HF myocytes.

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Relation between the shortening magnitude and [Ca2+]i transient amplitude (A), between time to 50% relaxation and that to 50% decline of [Ca2+]i transient (B), and between time to 90% relaxation and that to 90% decline of [Ca2+]i transient (C), in myocytes from control (open circles; n=10) and HF (closed circles; n=7) dogs. Each point represents the average of four to six myocyte data obtained from each dog. There was a significant positive relation (P<0.01) between each parameter of shortening and that of the [Ca2+]i transient.

 
3.3 Myofibrillar Ca²⁺ sensitivity in intact myocytes
We examined myofibrillar Ca²⁺ sensitivity in intact myocytes by plotting the relation between [Ca²⁺]i transient amplitude at end-systolic cell length and the peak cell shortening magnitude in varied Ca²⁺ concentrations of bathing media [17]. Peak cell shortening magnitude increased in response to the increase of Ca²⁺ concentration in control and HF myocytes. Similarly, [Ca²⁺]i transient amplitude increased in response to Ca²⁺ concentration in the media. The relation between cell shortening and the [Ca²⁺]i transient was thus shown to be linear. Before comparing myofibrillar Ca²⁺ sensitivity between control and HF, we confirmed that this relation was capable of detecting a change in Ca²⁺ sensitivity by exposing myocytes to EMD57033 (1 µmol/l) and BDM (5 mmol/l) (data not shown). To make direct comparison of myofilament Ca²⁺ sensitivity between control and HF, cell shortening and [Ca²⁺]i transient were examined over common (overlapping) ranges of shortening by exposing myocytes to a range of perfusate Ca²⁺ concentration of 0.8, 1.25, 2.5 and 5 mmol/l. Resting [Ca²⁺]i did not change significantly in control myocytes (222±4, 229±8, 226±8, and 228±8 nmol/l) when Ca²⁺ concentration in the bathing medium was 0.8, 1.25, 2.5 and 5 mmol/l, respectively. Similarly, in HF myocytes, resting [Ca²⁺]i did not change significantly (186±23, 184±21, and 185±23 nmol/l) when external Ca²⁺ was 1.25, 2.5 and 5 mmol/l, respectively. Peak cell shortening magnitude to [Ca²⁺]i transient amplitude at end-systolic cell length coordinates from HF myocytes lay along a relation with coordinates from control myocytes (P=NS by ANOVA). The slope and y-intercept of this relation from the averaged data were comparable between control and HF, suggesting that the myofilament Ca²⁺ sensitivity was preserved normal in HF myocytes (Fig. 3).

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Peak cell shortening magnitude to [Ca2+]i transient amplitude at the end-systolic cell length relation of the average data from control (open circles; n=10) and HF (closed circles; n=7) dogs during exposure of myocytes to the varying concentrations of bathing Ca2+ (0.8 to 5 mmol/l). There was no significant shift in the slope and the y-intercept of the correlation between control and HF.

 
Myocytes isolated from control and HF hearts were tetanized in the presence of ryanodine, producing steady-state levels of cell shortening and [Ca²⁺]i. Both cell shortening and [Ca²⁺]i transient reached a plateau level. As [Ca²⁺]o was increased, a progressive increase occurred in the magnitude of the steady-state levels of [Ca²⁺]i along with the steady-state cell shortening level, which increased from 0.625 to 20 mmol/l but reached a plateau at 15–20 mmol/l of [Ca²⁺]o. The steady-state cell shortening–[Ca²⁺]i relation was superimposable between control and HF (Fig. 4). As shown in Table 3, the maximal Ca²⁺-activated shortening (normalized to rest cell length), pCa₅₀, and Hill coefficient were comparable between control and HF (P=NS).

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 The relationship between steady-state cell shortening and [Ca2+]i in control (open circles; n=7 cells) and HF cells (closed circles; n=5 cells) at varied [Ca2+]o concentrations. Cell shortening magnitude was normalized to maximal Ca2+-activated shortening. The steady-state cell shortening–[Ca2+]i relation was superimposable between control and HF.

 

View this table: [in this window] [in a new window]   Table 3 Comparison of Hill parameters in intact and skinned myocyte preparations between control and HFa

 
3.4 Myofilament Ca²⁺ sensitivity in skinned myocytes
Another approach to assess myofibrillar Ca²⁺ sensitivity was based on the tension–pCa relation of skinned myocyte preparations. Before comparing this parameter between control and HF, we confirmed that this analysis was capable of detecting a change in myofibrillar Ca²⁺ sensitivity if it were present. EMD57033 (1 µmol/l) resulted in a leftward shift of the tension–pCa relation with a significant increase of pCa₅₀ from 6.11±0.06 to 6.51±0.03 (P<0.05). In contrast, isoproterenol (1 µmol/l) shifted the pCa–tension relation to the right, resulting in a small but significant decrease of pCa₅₀ from 6.02±0.03 to 5.93±0.04 (P<0.05). These results indicated that this analysis was capable of detecting the changes in myofibrillar Ca²⁺ sensitivity in myocyte preparations.

Tension–pCa relations were determined in skinned cells obtained from control (n=18 cells from ten dogs) and HF (n=13 cells from seven dogs). The average segment length of cells utilized for these determinations (the length between needles) was comparable (127±5 vs. 127±3 µm, P=NS) and the sarcomere length in a pCa 9.0 solution was also comparable (2.08±0.05 vs. 1.99±0.04 µm, P=NS). The maximum isometric tension of myocytes did not differ between control and HF (33±2 vs. 38±2 mN/mm², P=NS). The extent of internal shortening during the experiment was no more than 10% of the initial sarcomere length and did not differ between the two groups. Maximal active tension at the end of the experiments was 99±12% of the initial value in control and 93±4% in HF (P=NS), which indicated that the myocyte preparations were capable of maintaining maximum tension throughout the protocol. Fig. 5A shows the comparison of mean tension–pCa relationships for control and HF myocytes and the same data were linearized by using the Hill transformation (Fig. 5B). pCa₅₀ was 5.96±0.02 in control and 5.97±0.07 in HF (P=NS) and Hill coefficient was comparable (1.86±0.11 vs. 1.89±0.13, P=NS), which indicated that there was no reduction of myofilament sensitivity to Ca²⁺ in HF myocytes (Table 3). When sarcomere length was increased from 1.83±0.02 to 2.22±0.01 µm, pCa₅₀ increased from 5.97±0.04 to 6.20±0.08 µm (P<0.05) in control myocytes (Table 4). A similar length-dependent increase in the Ca²⁺ sensitivity of tension was noted in HF myocytes (pCa₅₀; 6.07±0.04 at short sarcomere length vs. 6.25±0.10 at long sarcomere length, P<0.05). Importantly, the mean change in pCa₅₀ values between short and long sarcomere lengths was comparable between control and HF (0.24±0.09 vs. 0.17±0.07, P=NS). The Hill coefficient was not altered when sarcomere length was changed.

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Cumulative tension–pCa plots (A) of skinned myocytes isolated from control (open circles; n=18 cells from ten dogs) and HF (closed circles; n=13 cells from seven dogs) and their Hill transformation (B). Both pCa50 and Hill coefficients for these relationships were comparable between control and HF.

 

View this table: [in this window] [in a new window]   Table 4 Effects of sarcomere length on the pCa—tension relation of skinned myocytea

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The present study demonstrates that (1) chronic rapid ventricular pacing produced contractile dysfunction at the isolated myocyte level in association with a parallel decrease in the peak systolic [Ca²⁺]i transient. (2) There was a significant positive correlation between the extent of myocyte shortening and the amplitude of [Ca²⁺]i transient. (3) In contrast, sensitivity of myofilament to Ca²⁺ in HF did not differ from that in control in intact as well as skinned myocyte preparations. These results indicate that the decrease of Ca²⁺ availability to the myofilaments, rather than the impairment of myofilament sensitivity to the cytoplasmic Ca²⁺, contributes to the abnormal contractile performance in the failing heart.

Peak systolic [Ca²⁺]i level in HF myocytes was lower than that in normal cells, which was consistent with previous studies [2–5]. In addition to this established observation, however, the new finding is that there is a significant positive correlation between the parameters of magnitude and duration of contractility and those in [Ca²⁺]i transient from HF myocytes (Fig. 2). Even though a close correlation does not prove a causal relationship, the changes in [Ca²⁺]i transient are sufficient to explain the contractile defects of failing myocytes and a decreased Ca²⁺ availability could be the mechanism responsible for the contractile dysfunction in this model of HF.

The mechanisms involved in the alterations in [Ca²⁺]i transient have not been well established. Recent work from this laboratory demonstrated that SR Ca²⁺-ATPase mRNA and Ca²⁺ uptake by SR were depressed in the same animal model of HF and the mRNA expression levels were significantly correlated with myocyte contractile function parameters [12]. Therefore, the reduced magnitude and prolonged time course of contraction as well as [Ca²⁺]i transient in HF were due to the abnormal Ca²⁺ uptake mechanisms by the SR. It is conceivable that reduced Ca²⁺ uptake by the SR should lead to smaller Ca²⁺ stores in the SR to be released during each contraction. It seems unlikely that the reduction in Ca²⁺ current accounts for the decreased [Ca²⁺]i transient observed in this study since the previous studies demonstrated that L-type Ca²⁺ channel function assessed with voltage clamp was not changed [23] and the properties of dihydropyridine bindings were not altered [24]. Further, our recent study demonstrated that the functional significance of the Na⁺/Ca²⁺ exchanger in the efflux of Ca²⁺ from the cytosol might be minor in this model of HF [12], even though the mRNA levels of the Na⁺/Ca²⁺ exchanger have been reported to be upregulated in the end-stage human failing hearts [25].

The second part of our study focused on the alterations in the sensitivity of myofilaments to Ca²⁺ in HF myocytes. Previous studies have yielded conflicting results concerning this important issue [2,3,6,7,9–11,26,27], which might result from the differences in the experimental conditions. The present study indicated that the normal sensitivity of myofilaments to Ca²⁺ was preserved in skinned HF myocyte preparations. The validity of our method was confirmed by the similarity of our Ca²⁺-activated maximal tension values (30 mN/mm²) with those reported previously in rat (31 or 37 mN/mm²) [8,28], canine (26 mN/mm²) [10], and human (30 mN/mm²) [11] and the proper response of the tension–pCa relation to the pharmacological intervention to alter myofibrillar Ca²⁺ sensitivity. Further, to exclude the possibility that the differences in loading conditions may affect the Ca²⁺ sensitivity [27,29], we measured the tension–pCa relation at both short and long sarcomere lengths. Ca²⁺ sensitivity of isometric tension was length-dependent and, importantly, there was no difference in Ca²⁺ sensitivity at either sarcomere length between control and HF (Table 4). These results suggested that there was no difference in myofilament Ca²⁺ sensitivity between control and HF even at a longer sarcomere length, which could maximize the differences in Ca²⁺ sensitivity if present. However, the direct measurement of myofilament Ca²⁺ sensitivity in the skinned preparation might be less physiological, which has been suggested by the alteration of Ca²⁺ sensitivity due to the skinning process itself (possibly by the phosphorylation of contractile proteins or alterations of crossbridge kinetics) [30]. Therefore, we also assessed myofibrillar Ca²⁺ sensitivity in intact myocyte preparations by using the contraction–[Ca²⁺]i transient amplitude relation (Fig. 3) and also the steady-state cell shortening–[Ca²⁺]i relation (Fig. 4) and demonstrated it not to differ between control and HF. Taken together, the results based on three different approaches were consistent, which confirmed that normal myofibrillar Ca²⁺ sensitivity was preserved in HF myocytes.

It should be acknowledged that, in contrast to our results, Wolff and his coworkers demonstrated that Ca²⁺ sensitivity of isometric tension was increased in failing hearts from canine tachycardia-induced HF [10] as well as from end-stage human HF [11] by measuring the isometric tension development in the myofibrillar preparations. The discrepancy between the present study and those by Wolff et al. might have resulted from the differences in the methods to prepare skinned myocytes. They employed myocyte-sized myofibrillar preparations obtained from small frozen ventricular biopsies, which might cause a possible sampling bias of selecting relatively ‘normal’ myocytes from failing hearts due to a very small number of successful preparations per experiment. Indeed, the cellular size of failing myocytes in their study did not differ from that of normal cells [10], which was in contrast to the cellular remodeling known to occur in this model of HF [31]. In addition, the use of powerful detergents such as Triton X-100 to permeabilize plasma membrane might produce the loss of cytosolic factors such as phosphatases and ions which might influence the binding of Ca²⁺ to myofibrils.

Several potential limitations should be acknowledged in this study. First, the differences in the effects of cell isolation process between control and HF could be responsible for the abnormalities in cell shortening and [Ca²⁺]i transient. However, it is unlikely since the isolation procedures were the same and both the yield of rod-shaped myocytes and the ratio of cells responding to electrical field stimulation to all cells did not differ between two groups of cells. In addition, maximal Ca²⁺-activated force in skinned myocytes was also comparable. Second, even though our methods of Ca²⁺ sensitivity measurement were well validated by the results that the pharmacological intervention to alter myofibrillar Ca²⁺ sensitivity could modulate the slope of [Ca²⁺]i -shortening relation or pCa–tension relation towards an appropriate direction, it should be recognized that they have limits of resolution like all other physical measurements. Therefore, the possibility that small but undetected changes in Ca²⁺ sensitivity may be produced by HF cannot be excluded absolutely.

Using both intact isolated and skinned myocyte preparations, this study presented evidence to suggest that a potential defect in myocyte contractile function following the development of pacing-induced HF is a reduction in Ca²⁺ availability to the myofilaments, rather than the inherent defects in myofilament sensitivity to Ca²⁺. There are two important findings in this study. First, we performed the parallel analysis of Ca²⁺ availability to the myofilaments and their sensitivity to Ca²⁺ in myocytes isolated from the single heart. Second, myofilament Ca²⁺ sensitivity was examined in both intact and skinned myocyte preparations isolated from the single heart, in which the cellular contractility had been characterized. Importantly, our myofilament Ca²⁺ sensitivity data were consistent irrespective of the types of preparations examined. Accordingly, the results of this study suggest that it is not necessary to implicate the myofilament Ca²⁺ sensitivity to explain the contractile abnormalities observed for our experimental model of HF. This in turn supports the notion that the therapy to correct the contractile dysfunction of the failing heart can rationally be directed to improving decreased availability of Ca²⁺ to the myofibrils.

Time for primary review 23 days.

	Acknowledgements

 
This work was supported in part by grants from the Ministry of Education, Science and Culture (No. 07266220, 08258221, 09670724).

Presented in part at the 70th Scientific Session of the American Heart Association, Orlando, Florida, November 9–12, 1997 and published in abstract form (Circulation 1997; 96 [Suppl I]: I–137).

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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