Cardiovascular Research

Cardiovascular Research 1997 33(1):88-97; doi:10.1016/S0008-6363(96)00187-3
© 1997 by European Society of Cardiology

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Copyright © 1997, European Society of Cardiology

Effects of inhibition of sarcoplasmic reticulum calcium uptake on contraction in myocytes isolated from failing human ventricle

K Davia, C.H Davies^a and S.E Harding^*

Cardiac Medidine, National Heart and Lung Institute, Imperial College, London SW3 6LY, UK

Received 29 February 1996; accepted 21 August 1996

	Abstract

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

 
Objectives: There has been debate regarding the level of sarcoplasmic reticulum (SR) Ca²⁺ ATPase protein in heart failure. We have used the SR Ca²⁺ ATPase inhibitor thapsigargin to investigate the functional contribution of this uptake system to contraction and relaxation in myocytes from failing and non-failing human ventricle. Methods: Myocytes were isolated from 28 failing and 18 non-failing ventricles and stimulated at 0.2 Hz, 32°C in Krebs-Henseleit solution. Contraction amplitude and speed were compared before and after treatment with 1 µmol/l thapsigargin for 20 min to deplete SR Ca²⁺ stores. Results: Initial beat duration was longer in myocytes from failing hearts. Addition of thapsigargin significantly prolonged the beat in myocytes from both groups, but the increase was greater in non-failing hearts (beat duration increased by 0.79 ± 0.12 s in myocytes from non-failing hearts compared with 0.37 ± 0.12 s in those from failing, P < 0.02). The contraction amplitude increased at high stimulation frequencies in myocytes from non-failing hearts (from 2.6% shortening at 0.1 Hz to 4.6% at 1 Hz, P < 0.001, n = 9), but not in those from failing hearts (1.8% at 0.1 Hz compared with 1.7% at 1 Hz, n = 5). Thapsigargin abolished the positive staircase in the non-failing, but had no effect in the failing group. Contraction amplitude following a rest interval was significantly depressed relative to steady-state levels in myocytes from the non-failing hearts (44.8 ± 10.3% at 3 min), but not in failing (102 ± 18%, P < 0.01 compared to non-failing at 3 min). Following thapsigargin treatment, there were no longer significant differences between failing and non-failing myocytes in the time course of the beat, frequency response or post-rest behaviour. Conclusion: The differences between myocytes from failing and non-failing hearts were reduced by inhibition of SR function. These results are consistent with the hypothesis that the initial differences had been due to decreased SR Ca²⁺ uptake.

KEYWORDS Human, ventricular myocytes; Relaxation; Sarcoplasmic reticulum; Heart failure; SR Ca-ATPase; Thapsigargin

	1. Introduction

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

 
Heart failure is a disease of diastolic as well as systolic dysfunction. Diastolic dysfunction can be observed in the absence of systolic problems in about 30% of heart failure patients [1]. Studies from isolated muscle [2] or single cells[3] show prolonged Ca²⁺ transients implying a defect in diastolic Ca²⁺ removal which could underlie poor relaxation in vivo. The two main systems for Ca²⁺ removal are the sarcoplasmic reticulum (SR) and Na⁺/Ca²⁺ exchanger. Studies have shown that SR Ca²⁺ ATPase mRNA is reduced [4–7] and Na⁺/Ca²⁺ exchanger mRNA is increased [6] in failing myocardium; however, there is debate about how this translates to protein levels and function. Experiments with contracting muscle strips showed that the decrease in amount of SR Ca²⁺ ATPase protein correlated with depression of the force-frequency response in failing human myocardium [8]. Other authors have also shown SR Ca²⁺ ATPase protein levels and Ca²⁺ uptake activity to be impaired in membrane preparations from failing human hearts [9–11]. However, there is disagreement in this area, since Movsesian et al. have not detected any decrease in SR Ca²⁺ ATPase protein levels in either crude or purified membranes from failing ventricle[12]. Results from this type of experiment are potentially confounded by the dilutional effects of other cell types [13, 14].

Since there is some uncertainty regarding levels of actual SR protein remaining in heart failure, we have investigated whether the SR is functionally impaired in diseased myocardium. We used a prolonged exposure to the selective SR Ca²⁺ ATPase inhibitor thapsigargin to remove the SR contribution to the beat [15, 16]. Experiments were done on isolated myocytes to avoid the problems of necrosis [17] and fibrosis [18] associated with intact failing myocardium, and also because thapsigargin penetrates poorly into muscle strips [19]. We compared contraction and relaxation with increasing frequency of stimulation in the presence of thapsigargin in myocytes from patients with and without heart failure. Contractile performance following complete blockade of the SR Ca²⁺ uptake allowed us to assess its relative importance to contraction and relaxation in the normal and diseased state, and also to observe the role of potential compensatory mechanisms such as the Na⁺/Ca²⁺ exchanger.

To investigate SR function further we exposed the myocytes to increasing periods of rest [20]. The amplitude of the first post-rest beat is thought to be a measure of the Ca²⁺ remaining in the SR [21]. Post-rest beats were compared between myocytes isolated from failing and non-failing human left ventricles: these experiments were repeated in the presence of thapsigargin.

Removal of the SR contribution to contraction and relaxation abolished the differences between myocytes from failing and non-failing hearts, supporting the hypothesis that SR dysfunction is present in human heart failure.

	2. Methods

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

 
2.1. Myocyte preparation from explanted hearts
Human ventricular myocardium was obtained from 28 patients in end-stage heart failure and 3 donor hearts not used for technical reasons: details are in Table 1 and Table 2. Informed consent was obtained before the operation. The investigation conforms with the principles outlined in the Declaration of Helsinki. Left ventricular free wall (approximately 3–5 g) was transported in ice-cold cardioplegia solution (mmol/l) Na 131, K 5, Cl 111, Ca 2, lactate 29 and procaine 20, and the average transport time to the laboratory was 90 min. Myocytes were isolated as previously described [22]. As the tissue arrived, a sample of the left ventricle from the epicardial region (average weight 1 g) was removed, this was cut into chunks of approximately 1 mm³ using a razor array and was incubated at 35°C in 25 ml of a low calcium (LC) medium containing nitrilotri-acetic acid (NTA) as a calcium buffer. The composition of the LC medium was (mmol/l) Na 120, K 5.4, Cl 125.4, Mg 5, SO₄ 5, pyruvate 5, glucose 20, taurine 20, HEPES 10 and NTA 5, bubbled with 100% O₂. The pH was adjusted to 6.95 and the measured free [Ca²⁺] was 1–3 µmol/l. The medium was changed 3 times at 3 min intervals (12 min total). The chunks were then drained and transferred to LC without NTA and with 50 µmol/l calcium added with 4 U/ml Sigma type XXIV protease (pronase) at the same temperature for 45 min. The solution was shaken under an atmosphere of 100% oxygen throughout. Two further digests were undertaken using Sigma type V collagenase 1 mg/ml with or without Sigma hyaluronidase 0.5 mg/ml. The cell suspension was then filtered through 300 µm gauze to remove undigested tissue and the myocytes were pelleted by gentle centrifugation. The pellet was resuspended in preoxygenated LC without NTA.

View this table: [in this window] [in a new window]   Table 1 Data from heart failure patients

 

View this table: [in this window] [in a new window]   Table 2 Data from patients without heart failure (donors)

 
2.2. Myocyte preparation from biopsies
Myocytes were isolated from left ventricular biopsies as previously described [23]. Patients were selected with stable angina undergoing coronary artery surgery with normal systolic ventricular function (ejection fraction > 60%) as defined by left ventricular angiography. Patients were excluded if they had a history of myocardial infarction. The details of the patients used in this study are described in Table 3. Following the institution of cardiopulmonary bypass the heart was electrically fibrillated and prior to the initiation of cardioplegia a small biopsy of the left ventricular free wall was made with a number 11 blade. The average biopsy weight was 125 mg. There were no adverse effects from this procedure.

View this table: [in this window] [in a new window]   Table 3 Data from patients without heart failure (biopsies)

 
The sample was placed into an ice-cold LC medium for 30 min. The sample was then placed in a vibratome (Micro Cut 1200, Energy Beam Sciences) and cut into 400 µm sections before being placed in an ice-cold solution of protease (0.5 mg/ml) and collagenase (1.5 mg/ml) in LC solution but with the addition of 50 µmol/l calcium and without NTA. This was then warmed to 35°C for 30 min whilst being gently shaken in an atmosphere of 100% oxygen. This solution was then exchanged for one containing collagenase (1.0 mg/ml) and hyaluronidase (0.5 mg/ml) and incubated for a further 90 min. The supernatant was removed and the myocytes pelleted by gentle centrifugation before resuspension in LC without NTA but with the addition of 0.5 g/l bovine serum albumin and a final calcium concentration of 300 µmol/l (patients 1–6). These solutions were initially supplemented by the addition of butanedione monoxime (BDM) 30 mmol/l to prevent cutting injury, and insulin 0.1 U/l to aid glucose uptake for patients 1–5. However, since their addition did not appear to improve cell yield, they were omitted from the protocol. The cell yield obtained could be up to approximately 70% but was usually less than 10%. Results obtained from myocytes in the presence of the BDM and insulin were no different from those isolated without. All patients gave informed consent and the procedure was approved by the ethical committee of the Royal Brompton National Heart and Lung Hospital.

2.3. Myocyte contraction experiments
Myocytes were placed in a bath on an inverted microscope stage as previously described [24] in Krebs-Henseleit solution, composition (mmol/l) Na 145, Cl 124.7, K 5.9, Mg 0.97 SO₄ 0.97, H₂PO₄^– 1.2, HCO₃^– 25, glucose 11, calcium 1.0 and equilibrated with 95% O₂/5% CO₂. Myocytes were chosen for study on the basis of a number of criteria: (1) morphological appearance (rod-shaped, no large blebs or areas of hypercontracture), (2) sarcomere length > 1.60 µm, (3) no spontaneous contractions when unstimulated at 1 mmol/l Ca²⁺ and (4) steady contraction amplitude and diastolic length at a stimulation rate of 0.2 Hz (using a bipolar pulse).

Experiments were carried out at 32°C. Field stimulation was at a frequency of 0.2 Hz and contraction was monitored by a video edge-detection system. Calcium concentrations were those giving approximately 50–75% maximum contraction [22, 25](4–8 mmol/l). When contraction had reached a steady state, the data were downloaded through a serial interface and averaged for time-to-peak contraction (TTP) and times to 50% (R50) and 90% (R90) relaxation. Once a steady state had been achieved, field stimulation was stopped for 10 s, 30 s or 3 min and the size of the first 10 post-rest beats was compared with the steady state. These recordings were then repeated after a 20 min exposure of the contracting cell to 1 or 3 µmol/l thapsigargin. In another series of experiments myocytes were stimulated at increasing frequencies as previously described [23], before and after 20 min exposure to 1 µmol/l thapsigargin.

2.4. Statistical analysis
When data were obtained from more than one myocyte from each preparation, the results were pooled, so that values are mean ± sem where n = patients (except for Fig. GR7 where results from individual myocytes are shown). Results were compared by paired or unpaired t-test, with correction for unequal variances where appropriate. Data were tested for normality, and where this was not confirmed (R50 values for non-failing myocytes), the non-parametric Mann-Whitney test was used instead of the two-sample t-test. Analysis of covariance and linear regression were performed on pooled data using the MINITAB program (Pennsylvania).

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. GR7 Contraction amplitude of beats following 3 min rest in thapsigargin-treated (1 µmol/1) myocytes from 10 non-failing () and 7 failing () hearts. Amplitude is expressed as a percentage of the previous steady-state contraction (0.2 Hz), which is indicated by the dotted line.

 

	3. Results

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

 
3.1. Contraction characteristics of human left ventricular myocytes
At a stimulation frequency of 0.2 Hz, there is little difference in contraction amplitude between myocytes from failing and myocytes from non-failing ventricle, at any Ca²⁺ concentration [22]. However, in the present study time-to-peak contraction (TTP) was significantly slowed in myocytes from failing (n = 26) compared to non-failing (n = 18) hearts (0.39 ± 0.03 vs 0.30 ± 0.02 s, P < 0.02), as was time-to-50% relaxation (R50) (0.23 ± 0.01 vs 0.18 ± 0.02 s, P = 0.02). The more variable time-to-90% relaxation (R90) was not significantly different in myocytes from the failing hearts compared to those from the non-failing (0.59 ± 0.04 vs 0.47 ± 0.06 s). TTP and R50 were not obtained from 2 patients in the failing group because the contraction amplitude of the myocytes was too small to allow accurate measurement of these parameters. The changes are similar to those we have previously reported in a separate series of patients [25].

3.2. Effect of thapsigargin on contraction characteristics
Myocytes contracting in Ca²⁺ concentrations around the EC₅₀ to EC₇₅ values (4–8 mmol/l) were superfused with Krebs-Henseleit solution containing 1 µmol/1 thapsigargin for 20 min. The concentration and exposure time required were greater than those used for guinea-pig myocytes where 500 nmol/l for 3–4 min was shown to abolish the SR Ca²⁺ release produced by cooling contractures[26]. Addition of ryanodine in the presence of thapsigargin yielded no further effect, confirming the level of thapsigargin as maximally effective (data not shown).

The effect of thapsigargin on contraction amplitude was variable between myocytes, but there was no consistent decrease in amplitude of contraction of myocytes from either the failing or the non-failing myocardium at 0.2 Hz (Table 4). A slight but consistent decrease in diastolic length was seen in cells from the non-failing group treated with 1 µmol/l thapsigargin, but not in the failing group (Table 4).

View this table: [in this window] [in a new window]   Table 4 Cell length and contraction amplitude of myocytes in the presence and absence of thapsigargin

 
There was, however, a significant prolongation of the beat in myocytes from both the non-failing and the failing hearts in the presence of thapsigargin, with the myocytes from the non-failing hearts more severely affected. Total duration of the beat (time from the start of contraction to 90% relaxation, TTP + R90) was increased by 0.79 ± 0.12 s (109 ± 19%, P < 0.001) in myocytes from non-failing hearts compared with 0.37 ± 0.12 s (45 ± 18%, P < 0.05) in those from failing hearts. The increase in duration was significantly greater in the non-failing than the failing group (P < 0.02).

Fig. GR1 illustrates the mean data of the effect of thapsigargin on the time course, divided into TTP, R50 and R90, with sample traces in Fig. GR2. Thapsigargin prolonged TTP by 0.11 ± 0.04 s (34.9 ± 10.2%, P < 0.05, n = 10) in the non-failing compared to 0.07 ± 0.02 s (17.2 ± 6.8%, P < 0.02, n = 12) in the failing myocytes. R50 was increased by 0.25 ± 0.07 s (221 ± 79%, P < 0.05, n = 10) in non-failing compared to 0.13 ± 0.04 s (66 ± 24%, P < 0.02, n = 12) in failing, and R90 by 0.67 ± 0.09 s (187 ± 44%, P < 0.01, n = 9) in non-failing compared to 0.29 ± 0.13 s (71 ± 33%, P = n.s., n = 8) in failing myocytes. In several cases R90 could not be measured with sufficient accuracy before addition of thapsigargin. The increases in TTP and R50 considered separately were not significantly different between groups, but the increase in R90 was greater in myocytes from non-failing than from failing hearts (P < 0.05).

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. GR1 Times-to-peak contraction (TTP) and from peak to 50% relaxation (R50) and 90% relaxation (R90) in myocytes from 12 failing (F) and 10 non-failing (NF) human hearts in the absence (–) and presence (+) of 1 µmol/l thapsigargin. Significantly different from before thapsigargin, paired t-test:# P < 0.05, * P < 0.02, ** P < 0.01, *** P < 0.001.

 

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. GR2 Sample traces (average of 6 beats) showing the effect of 1 µmol/l thapsigargin on the time course of the beat in myocytes from non-failing and failing human hearts. Solid lines are in the absence and dotted lines in the presence of thapsigargin. Contraction amplitude has been normalised to that in the absence of thapsigargin to show time course changes. The myocyte from non-failing heart was 129 µm long with a sarcomere length of 1.90 µm, and the contraction amplitudes before and after thapsigargin were 4.3 and 6.6% cell length, respectively. The myocytes from failing heart was 106 µm long with a sarcomere length of 1.98 µm, and the contraction amplitudes before and after thapsigargin were 6.0 and 5.5% cell length, respectively. Both myocytes were contracting stably in 8 mmol/l Ca2+.

 
Following functional SR removal with thapsigargin we no longer found a significant difference in TTP, R50 or R90 between the non-failing and failing hearts. Final values (non-failing vs. failing) were: TTP 0.41 ± 0.05 vs. 0.48 ± 0.06 s; R50 0.42 ± 0.06 vs 0.34 ± 0.04 s; and R90 1.18 ± 0.11 vs 0.98 ± 0.11 s. The final beat durations (TTP + R90) were 1.59 ± 0.15 s for myocytes from non-failing and 1.47 ± 0.13 s in myocytes from failing hearts.

3.3. Effect of thapsigargin on frequency dependence of contraction amplitude
We have previously shown that myocytes from non-failing human hearts show a positive staircase, with markedly increased contraction amplitude at high stimulation frequencies (1–1.5 Hz) compared to low (0.1–0.2 Hz) [23](Fig. GR3). This is shown in the top panel of Fig. GR3, for a separate group of cells contracting in 2 mmol/l Ca²⁺. After thapsigargin treatment (1 µmol/l, 20 min) the response to increasing frequency of stimulation was significantly depressed (P < 0.001, ANCOVA), although the amplitude of contraction at 0.1 Hz was not significantly altered (2.6 ± 0.4% before and 3.4 ± 0.7% after thapsigargin, n = 9). This is shown in the lower panel of Fig. GR3, with amplitude expressed as a percentage change compared to that at 0.1 Hz. A substantial proportion of myocytes from non-failing hearts failed to follow pacing above 0.5 Hz in the presence of thapsigargin: the number of cells remaining is indicated on the figure.

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. GR3 Contraction amplitude with increasing stimulation frequency, as a percentage of the amplitude at 0.1 Hz, in the absence (upper panel) and presence (lower panel) of 1 µmol/l thapsigargin. Experiments were done in a paired manner on 9 cells from 9 non-failing hearts and 5 cells from 5 failing hearts in 2 mmol/l Ca2+. Contraction amplitudes (% cell shortening) at 0.1 Hz were 2.6 ± 0.4 before and 3.4 ± 0.7% after thapsigargin for non-failing and 1.8 ± 0.2 before and 1.9 ± 0.4% after thapsigargin for failing groups. After thapsigargin treatment some cells did not follow pacing above 0.5 Hz; numbers on the graph in brackets indicate the number of cells still able to contract at a given frequency. Curves were significantly different before and after thapsigargin treatment for the non-failing group (ANCOVA, P < 0.001) but not the failing.

 
Myocytes from failing hearts displayed a negative rather than positive staircase (Fig. GR3). Average amplitude at 0.1 Hz was 1.8 ± 0.2% cell shortening, not significantly different from non-failing. However, by the time that the frequency was increased to 1 Hz there was a depression of contraction in cells from failing hearts (1.7 ± 0.4%, n = 5) compared to non-failing (4.6 ± 0.7%, n = 9, P < 0.01). This has been described in detail previously [23]. Thapsigargin had no significant effect on the frequency dependence of contraction in myocytes from failing heart.

Following thapsigargin treatment, frequency responses were superimposable for myocytes from failing and non-failing hearts, where data could be obtained (Fig. GR3). However, myocytes from failing hearts withstood thapsigargin treatment notably better than the non-failing, with all of the cells following pacing to 1 Hz.

3.4. Post-rest experiments
The contraction amplitudes of the initial post-rest beats, expressed as a percentage of the previous steady state (0.2 Hz), were significantly different between left ventricular myocytes from patients with and without heart failure (Fig. GR4). With increasing intervals, myocytes from non-failing hearts displayed a progressive decay in contraction amplitude, with B1 (the first post-rest beat) falling to 44.8 ± 10.3% (n = 18) of steady state following 3 min rest. Myocytes from failing hearts exhibited a post-rest contraction amplitude similar to pre-rest levels (Fig. GR4, and sample traces in Fig. GR5). The amplitude following 3 min rest averaged 102 ± 18% (n = 26) of steady state.

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. GR4 Contraction amplitude of beats following 10 s, 30 s or 3 min rest in myocytes from 18 non-failing and 26 failing human hearts. Amplitude is expressed as a percentage of the previous steady-state contraction (0.2 Hz), which is indicated by the dotted line. * P < 0.05 – P < 0.001 for differences with failing heart.

 

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. GR5 Sample traces showing the first beats following a rest interval in myocytes from non-failing and failing human hearts. Cell characteristics for non-failing were: 100 µm length, 1.91 µm sarcomere length, contracting in 4 mmol/l Ca2+, and failing 162 µm length, 1.80 µm sarcomere length, contracting in 6 mmol/l Ca2+. The frequency of stimulation was 0.2 Hz both before and after rest: the time base is expanded for the initial post-rest period.

 
There was also a significantly increased variability between cells from failing hearts (variance ratio non-failing vs. failing P < 0.01 for B1 at 3 min); Fig. GR6 illustrates the size of the first post-rest beat after 3 min rest in individual myocytes. Post-rest potentiation of contraction was shown in 10 out of 30 myocytes isolated from the failing hearts, but in 0 of 23 from non-failing. The relation between the occurrence of post-rest potentiation of contrction and prolongation of TTP, R50 and R90 was investigated for individual myocytes. Within the failing group, cells with the slowest R50 values had the largest post-rest contractions at 3 min (B1 vs. R50, P = 0.1; B2 vs. R50, P = 0.04, n = 30 cells).

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. GR6 Amplitude of the first beat after 3 min rest, as a percentage of the steady-state amplitude, in 23 individual myocytes from 18 non-failing hearts and 30 myocytes from 26 failing human hearts. The dotted line indicates the steady-state (0.2 Hz) contraction amplitude (100%).

 
3.5. Rest experiments in the presence of thapsigargin
With myocytes from non-failing hearts the contraction amplitude of the initial beat following 3 min rest was significantly increased by 1 µmol/l thapsigargin from 35.9 ± 4.7 to 55.9 ± 4.4% (P < 0.01, n = 10) of the previous steady state. Conversely, thapsigargin depressed the first post-rest beat in cells from failing heart, with the result that differences between myocytes from failing and non-failing hearts were abolished and restitution curves became superimposable (Fig. GR7). In contrast to the findings on beat duration and frequency response, thapsigargin treatment did not convert the non-failing cells to the same post-rest contraction pattern as failing, but brought both to an intermediate level.

3.6. Patient characteristics
The size of the post-rest beat and the effect of thapsigargin were investigated relative to the aetiology of disease and the drug treatment of the patient. There was no significant difference between the responses of cells from patients with ischaemic heart disease and those with idiopathic dilated cardiomyopathy. Numbers of patients with mitral valve disease or congenital abnormalities were too low to permit analysis, but there was no obvious difference in these groups. Nor was there any significant difference in response between myocytes isolated from non-failing hearts by the biopsy method and those isolated from larger pieces of donor hearts.

There was also no significant association between the responses of the myocytes and the use of ACE inhibitors, digoxin, β-blockers, inotropes, diuretics, nitrates, antiar-rhythmic or anticoagulant agents or lipid-lowering drugs in the patients. Patients who have been taking Ca²⁺-antagonists in the failing group had significantly lower post-rest beats (B1 at 3 min was 38.1 ± 13% (n = 4) of steady state compared with 123 ± 21% (n = 21) in patients not on these agents, P = 0.03). It will be interesting to see whether this observation is confirmed in larger groups of patients. In the non-failing group B1 at 3 min was 30.2 ± 6.9% of steady state in patients on Ca²⁺ antagonists (n = 9) compared with 65 ± 24% in the remainder (n = 7) (P = n.s.).

	4. Discussion

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

 
The major findings from this study are that abolition of SR function prolongs beat duration and reduces the differences in contraction times, frequency dependence and post-rest beat amplitude between myocytes from failing and non-failing human hearts.

We have used thapsigargin [15, 16], a potent inhibitor of the SR Ca²⁺ ATPase, to investigate whether the abnormal contraction and relaxation we had observed in myocytes from failing human hearts could be reproduced by preventing SR Ca²⁺ uptake in non-failing cells. Thapsigargin was applied to contracting myocytes for 20 min, at a concentration double that required to abolish cooling- or caffeine-induced SR Ca²⁺ release within 3 min in animal myocytes. Since further application of thapsigargin or ryanodine had no additional effect on the beat, we consider that the SR contribution to contraction and relaxation had been abolished under these conditions. The subsequent contractile performance showed a markedly prolonged relaxation in myocytes from failing and non-failing hearts. This effect was significantly more pronounced in myocytes from patients without heart failure, suggesting a greater dependence on an unimpaired SR for relaxation in control conditions. When challenged with sudden inhibition of SR function, the Na⁺/Ca²⁺ exchanger and sarcolemmal pump may be insufficient to compensate and prolongation of the calcium transient results. Despite the fact that the effect of thapsigargin was less marked in myocytes from the failing hearts, inhibition of SR function did prolong the beat. This demonstrates that SR function is still contributing to calcium homeostasis in heart failure.

There was no longer a significant difference in relaxation times between myocytes from failing and non-failing hearts in the presence of thapsigargin. This is consistent with the hypothesis that initial differences were related to SR function. It was noted that the failing and non-failing could even show the reverse effect, with the non-failing relaxing more slowly than the failing following thapsigargin (see Fig. GR2).

Contraction amplitude was not affected by thapsigargin at low stimulation frequencies in either the failing or non-failing groups. This is consistent with results in guinea-pig myocytes, where it has been shown that ryanodine (which increases SR Ca²⁺ loss), but not thapsigargin, can reduce the size of the beat [27]. It has been suggested that Ca²⁺ uptake by the SR curtails the rise in Ca²⁺ at the contractile apparatus. Inhibition by thapsigargin prevents this effect and so extends the time for which the myofilaments see increased Ca²⁺: at low frequencies this lengthening of TTP balances the loss of Ca²⁺ released from the SR. In support of this, application of thapsigargin prevents or reverses the negative inotropic effect of ryanodine[27]. At higher frequencies of contraction the (non-failing) human myocytes were more dependent on the Ca²⁺ released from the SR for contraction, as thapsigargin now had a pronounced negative inotropic action.

In myocytes from failing hearts the positive staircase with increasing frequency was absent (as we have previously described [23]), and application of thapsigargin had little effect. This would support the hypothesis that SR function is compromised in the cells isolated from failing myocardium.

Again, the difference between myocytes from failing and non-failing hearts was abolished after thapsigargin treatment, suggesting that the original difference in frequency response had been SR-mediated. This supports the evidence from whole muscle studies where changes in frequency response have been attributed to alterations in SR Ca²⁺ handling [11, 28, 29], and correlated to decreases in the level of the SR Ca²⁺ ATPase protein [8]. Also as before, myocytes from non-failing hearts were markedly less able to tolerate thapsigargin, with a majority failing to complete a frequency run. Taken together with the relaxation data above, this suggests that there has been some change in the myocytes from the failing hearts that partially offsets the SR loss. These observations could lend tentative support to the hypothesis that Na⁺/Ca²⁺ exchange up-regulation in human heart failure partly compensates for SR dysfunction [6, 30].

Myocytes from the non-failing hearts demonstrated decay of the post-rest beats which became more pronounced with increasing rest intervals, as might be predicted from the frequency-response curve. This result contrasts with the findings of Pieske et al. who describe post-rest potentiation with isolated ventricular muscle strips from non-heart-failure patients [31]. However, the different experimental conditions used may account for these findings. The steady-state stimulation rate of 1 Hz used by Pieske et al, compared with 0.2 Hz used in this study, may have resulted in a transient SR Ca²⁺ load which was maintained over short rest intervals. We have found that high-frequency steady-state stimulation produces a similar effect in human ventricular myocytes (data not shown). Evidence from rat heart experiments has shown that under identical experimental conditions there is no difference in post-rest beat amplitude between ventricular strips and single myocytes[20].

Post-rest decay of contraction can be a consequence of calcium loss from the SR during rest. When repeated in the presence of thapsigargin the extent of decay was reduced, but not eliminated, suggesting that part but not all is SR-related.

Myocytes from failing hearts displayed much less post-rest decay than myocytes from non-failing hearts. This could be due to a less leaky SR, either because of increased membrane stability or less frequent spontaneous openings of the SR release channel, although recent evidence has suggested that Ca²⁺ homeostasis during periods of rest may be more dependent on sarcolemmal ion fluxes than SR Ca²⁺ leak [32]. Alternatively, if the SR contribution to steady-state contraction is reduced, then contraction amplitude, pre- and post-rest, may primarily reflect Ca²⁺ influx. However, myocytes from failing hearts also demonstrated a varying degree of post-rest potentiation. Thapsigargin treatment reduced post-rest potentiation, suggesting an SR-related phenomenon. This type of potentiation is similar to that seen in rat myocytes where the Na⁺/Ca²⁺ exchanger spends a greater proportion of the cardiac cycle in calcium influx mode due to increased intracellular sodium [33]. We speculate that post-rest potentiation in human myocytes may be related to a change in sodium regulation because of alterations in the expression of the Na⁺/Ca²⁺ exchanger.

Comparison between myocytes from failing and non-failing hearts in the presence of thapsigargin demonstrated no significant difference in post-rest contraction behaviour. The residual post-rest decay was similar in both, which suggests that the non-SR rest-dependent mechanisms do not differ between failing and non-failing hearts.

	5. Conclusions

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

 
Prolongation of the beat and depressed frequency responses previously described in myocytes from failing human hearts could be reproduced in myocytes from non-failing hearts by inhibition of SR function. After thapsigargin treatment the performance of myocytes from non-failing hearts was not significantly different from failing hearts, where data could be obtained. This is supporting evidence that the original difference was SR-mediated. However, the post-rest potentiation often seen in failing cells is not mimicked by thapsigargin treatment in non-failing cells, which suggests alterations of Ca²⁺ homeostasis over and above a simple loss of functional SR Ca²⁺ uptake. It was also more difficult to obtain data at high stimulation frequencies from thapsigargin-treated, non-failing myocytes than from similarly treated failing cells, suggesting some compensatory change in heart failure which tends to minimise the loss of SR function.

	Acknowledgements

 
Kerry Davia was supported by Pfizer, and Crispin Davies by the British Heart Foundation. Sian Harding is a Wellcome Senior Lecturer. We are grateful for the assistance of the staff of Harefield and the Royal Brompton Hospitals for assistance with supply of tissue.

	Notes

 
^a Current address: Department of Cardiovascular Medicine, John Radcliffe Hospital, Oxford, UK.

^* Corresponding author. Tel. +44 171 352-8121, ext. 3311; Fax +44 171 823-3392; E-mail sian.harding @ic.ac.uk

	References

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