Reduced Contraction and Altered Frequency Response of Isolated Ventricular Myocytes From Patients With Heart Failure
Background Previous work has failed to demonstrate reduced maximal contraction of isolated ventricular myocytes from failing human hearts compared with nonfailing control hearts. The effect of alterations in stimulation frequency and temperature on the contraction of isolated ventricular myocytes has been investigated. Left ventricular myocytes were isolated from the hearts of patients with severe heart failure undergoing heart transplantation and compared with myocytes isolated from myocardial biopsies from patients with coronary disease but preserved left ventricular systolic function or from myocytes from rejected donor hearts.
Methods and Results Myocytes were exposed to either a maximally activating level of extracellular calcium at 37°C or to 2 mmol/L calcium at 32°C. There was no significant difference in the contraction amplitude between myocytes from failing and nonfailing hearts at 0.2 Hz. With increasing stimulation frequency, there was a reduction in contraction amplitude in cells from failing hearts relative to control hearts in both maximal calcium from 0.33 Hz (4.5% versus 6.6%) to 1.4 Hz (3.9% versus 8.8%) (ANCOVA, P<.001) and at 2 mmol/L calcium from 0.50 Hz (2.3% versus 3.5%) to 1.4 Hz (1.8% versus 3.9%) (ANCOVA, P<.001). The time to peak contraction and the times to 50% and 90% relaxation were prolonged in myocytes from failing hearts at stimulation rate of 0.2 Hz (P<.01), but only the time to 50% relaxation was prolonged at 1.0 Hz (P<.05).
Conclusions Reduced contraction, slowed relaxation, and impaired frequency response occurring at the level of the individual ventricular myocyte can be demonstrated in human heart failure. This demonstrates that disruption of myocyte function can contribute to both the systolic and the diastolic abnormalities that occur in the failing human heart.
There remains uncertainty as to the contractile function of the individual ventricular myocytes in the failing human heart. Our previous studies have demonstrated β-adrenoceptor desensitization1 as well as prolongation of TTP and R50 in myocytes isolated from patients with left ventricular failure.2 However, the maximum contraction amplitude has not been reduced in myocytes obtained from failing hearts compared with nonfailing control hearts.1
This surprising finding might be a reflection of either the true state of the remaining viable myocytes in human heart failure or merely inappropriate experimental conditions. As we recently reviewed,3 studies with papillary muscle and trabecular preparations have shown conflicting results, with some demonstrating reduced force of contraction in preparations from failing hearts4 5 and others unable to demonstrate a difference.6 7 As the studies that demonstrated a reduction in the force of contraction were in general those that used higher temperatures and stimulation frequencies, we examined the effects of these variables on the contraction amplitude of ventricular myocytes isolated from patients with normal and impaired systolic left ventricular function.
In addition, we have been concerned about the suitability of the hearts of brain-dead organ donors for use as normal control subjects in these experiments. We therefore investigated the use of small biopsy samples obtained from patients with normal systolic function undergoing coronary surgery to serve as control subjects.
Myocyte Preparation From Explanted Hearts
Human ventricular myocardium was obtained from 18 patients with severe cardiac failure secondary to ischemic heart disease (n=12), dilated cardiomyopathy (n=5), or end-stage valvular disease (n=1) at the time of transplantation (Tables 1⇓ and 2⇓). Informed consent was obtained before the operation. Myocytes were also isolated from the hearts of 2 patients without heart failure whose hearts were technically unsuitable for use as organ donors (Table 3⇓). Tissue was transported in ice-cold cardioplegia solution (131 mmol/L Na+, 5 mmol/L K+, 111 mmol/L Cl−, 2 mmol/L Ca2+, 29 mmol/L lactate, and 20 mmol/L procaine). Average transit time to the laboratory was 90 minutes, although in 4 patients (indicated by an asterisk in Tables 1⇓ and 2⇓) the average transit time was <5 minutes. Myocytes were isolated as previously described.1 A sample of the left ventricle (average weight, 1 g) was cut into chunks of approximately 1 mm3 with the use of a razor array and was incubated at 35°C in 25 mL of an LC medium containing NTA as a calcium buffer. The composition of the LC medium was (in mmol/L): NaCl 120, KCl 5.4, MgSO4 5, pyruvate 5, glucose 20, taurine 20, HEPES 10, and NTA 5, bubbled with 100% O2. pH was adjusted to 6.95, and the measured free [Ca2+] was 1 to 3 μmol/L. The medium was changed three times at 3-minute intervals (12 minutes 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 minutes. The solution was shaken under an atmosphere of 100% oxygen throughout the procedure. Two additional digests were undertaken using 1 mg/mL Sigma type V collagenase with or without 0.5 mg/mL Sigma hyaluronidase. 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.
Myocyte Preparation From Biopsies
We used a modification of our method for explanted hearts, incorporating some of the suggestions of Peeters et al.8 Fifteen patients were selected with stable angina who required coronary artery surgery but who had unimpaired systolic ventricular function (ejection fraction >60% as defined by left ventricular angiography) (Tables 4⇓ and 5⇓). Patients were excluded if they had a history of myocardial infarction. After the institution of cardiopulmonary bypass, the heart was electrically fibrillated, and before the instillation of cardioplegia, a small biopsy sample of the left ventricular free wall was taken with a No. 11 blade. The average biopsy weight was 125 mg. There were no adverse effects from this procedure.
The sample was placed into an ice-cold LC medium for 30 minutes. 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 minutes while 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 an additional 90 minutes. The supernatant was removed, and the myocytes were pelleted by gentle centrifugation before resuspension in LC without NTA but with the addition of 0.5 g/L BSA and a final calcium concentration of 300 μmol/L (patients 1 through 10). These solutions were supplemented by the addition of BDM 30 mmol/L for biopsy patients 1 through 5 and the addition of insulin 0.1 U/L for biopsy patients 1 through 6. All patients gave informed consent, and the procedure was approved by the Ethical Committee of the Royal Brompton National Heart and Lung Hospital.
Myocyte Contraction Experiments
Myocytes were placed in a bath on an inverted microscope stage as previously described9 in Krebs-Henseleit solution with a composition of (in mmol/L): NaCl 120, KCl 4.7, MgSO4 0.97, KH2PO4 1.2, NaHCO3 25, glucose 11, and 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 >1.72 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 biphasic pulse).
Experiments were performed in two groups; with the exception of transplant group A patients 8 and 9 and nonfailing group A patient 7, whose cells were examined in both groups, the two groups were mutually exclusive. Myocytes of group A patients were studied at 37°C in maximally stimulating Ca2+ (defined as the point at which no further increment in contraction occurred after an increase in the level of extracellular Ca2+). Myocytes from group B and those from the nonfailing donor hearts were studied at 32°C in 2 mmol/L Ca2+. The effect of a range of frequencies on contraction amplitude was then examined (0.1 to 1.42 Hz), following which the cell was again stimulated at 0.2 Hz to ensure internal consistency. Contraction was monitored using a video edge-detection system.9 Contraction was described either in terms of the systolic and diastolic sarcomere lengths (μm) or in terms of percent systolic shortening (systolic change in cell length/diastolic cell length). The TTP and R50 and R90 were measured from three consecutive contractions in myocytes in maximal Ca2+. The lower contraction amplitudes of myocytes in 2 mmol/L Ca2+ precluded the accurate measurement of these data in group B patients.
Significance was assessed on grouped data with the ANCOVA (or the general linear model where appropriate) and Student’s t test (for paired and unpaired samples as appropriate). Data from one to three cells for each patient were pooled before analysis. Values are expressed as mean±SEM.
Salts were obtained from Merck and were AnalaR grade except for KCl, taurine, and glucose, which were AristaR grade. AnalaR water was used for the low Ca2+ solutions and double distilled deionized water (MilliQ system) was used for the remainder. BDM was obtained from Sigma Chemical Co, and human insulin was obtained from Eli Lilly.
Basal Sarcomere Lengths
There was a nonsignificant trend toward longer sarcomere lengths under basal conditions (1 mmol/L Ca2+ and a stimulation rate of 0.2 Hz) of group A patients in myocytes from control subjects with nonfailing hearts (1.90±0.03 μm) than from patients with heart failure (1.82±0.03 μm, P=NS). This difference in diastolic sarcomere length became statistically significant in maximally activating Ca2+ (at 0.2 Hz: nonfailing, 1.88±0.03 μm; failing, 1.79±0.03 μm; P<.05) (Fig 1⇓, top). In group B patients, sarcomere lengths under basal conditions were not significantly different between nonfailing and failing hearts (nonfailing, 1.84±0.03 μm; failing, 1.84±0.02 μm) (Fig 1⇓, bottom). Because BDM was present for six of eight in the series A control subjects (37°C) but not for series B (32°C), we also examined the sarcomere lengths obtained during a parallel series of experiments performed at 32°C in which cells were prepared in the presence of BDM. In these experiments, there was no difference in the sarcomere lengths between cells from nonfailing (1.89±0.06 μm, n=13) and failing (1.90±0.04 μm, n=20 cells, P=NS) groups. Further experiments comparing the effects of vibratome versus razor sectioning in human atrial myocytes failed to detect any differences in sarcomere length between the two methods.
Contraction at 0.2 Hz
There was no significant difference in the contraction amplitude expressed in terms of percentage shortening of cells from failing and nonfailing hearts at 0.2 Hz in either maximal or 2 mmol/L Ca2+, and this finding is in accordance with our previous data obtained at 32°C in maximal Ca2+10 (Fig 2⇓). The reduced magnitude of the maximally stimulated contraction at 37°C compared with experiments performed at lower temperatures is also consistent with other studies.11 Maximal contraction in response to increasing calcium was obtained in myocytes from group A patients at 8.71±0.78 mmol/L Ca2+ in cells from control patients and 7.75±0.62 mmol/L in cells from patients with heart failure (P=NS).
Contraction in Maximal Calcium With Increasing Stimulation Frequency at 37°C (Group A Patients)
With increasing stimulation frequency, there was a progressive increase in the amplitude of contraction in myocytes from nonfailing hearts, whereas in those from failing hearts there was a decline. These markedly different responses to increases in stimulation rate were statistically significant overall (ANCOVA, P<.001) and at the individual frequencies of 0.33 (P<.05); 0.5 (P<.01); and 0.66, 0.80, 1.0, and 1.42 Hz (P<.001) (t tests) (Tables 6⇓ and 7⇓ and Fig 3⇓). The results obtained without the use of BDM in patients 6 and 7 (Table 4⇑) were similar in terms of the increased amplitude obtained with increasing stimulation frequency and in terms of the increased contraction compared with cells from patients with heart failure at higher frequencies. Systolic and diastolic sarcomere lengths decreased in parallel in response to increases in simulation rate for both failing and nonfailing hearts, but the changes in both were greater for cells from nonfailing hearts (Tables 6⇓ and 7⇓; ANCOVA, failing versus nonfailing P<.01 for both systolic and diastolic sarcomere lengths).
Contraction in 2 mmol/L Ca2+ With Increasing Stimulation Frequency at 32°C (Group B Patients)
Myocytes from 5 patients (2 with nonfailing and 3 with failing hearts) did not follow the driving frequency at 1.4 Hz. As with the experiments conducted in maximal calcium, there was a progressive increase in contraction amplitude in myocytes from nonfailing hearts with increasing stimulation rate (Tables 8⇓ and 9⇓ and Fig 4⇓). In contrast, there was an initial increase in shortening of myocytes from failing hearts with increasing stimulation frequency up to 0.5 Hz, although this was smaller than that seen with myocytes from nonfailing hearts (Fig 4⇓). However, at stimulation rates in excess of 0.5 Hz, there was a progressive decline in contraction amplitude. This divergence in the response to increasing stimulation frequency resulted in a significant depression of contraction amplitude in myocytes from patients with heart failure at higher stimulation rates (ANCOVA, failing versus nonfailing hearts, P<.001). Similar results were obtained in myocytes obtained from the nonfailing donor hearts (Table 3⇑). There was a progressive decrease in systolic sarcomere lengths in response to increasing stimulation rate in myocytes from nonfailing hearts in contrast to the flatter response in myocytes from patients with heart failure (failing versus nonfailing hearts, ANCOVA, P<.05) (Tables 8⇓ and 9⇓ and Fig 5⇓). There were small reductions (of the order of 1%) in diastolic sarcomere length with increasing stimulation rate in myocytes from both failing and nonfailing hearts, and these were significant by paired t test (P<.02). There was, however, no difference in diastolic shortening between the two groups (failing versus nonfailing ANCOVA, P=NS) (Figs 1⇑ and 5⇓).
Characteristics of Contraction and Relaxation
TTP and R50 and R90 were significantly prolonged in cells from failing hearts at a stimulation frequency of 0.2 Hz (P<.01) (Fig 6⇓, top). This is consistent with our previous observations at 32°C.2 At a stimulation rate of 1.0 Hz, only R50 achieved statistical significance (Fig 6⇓, bottom). This was due to a significant reduction in R50 and R90 in myocytes from failing hearts at 1.0 Hz compared with 0.2 Hz (P<.05) and to an increase in TTP in myocytes from nonfailing hearts (P<.05). R50 and R90 in myocytes from nonfailing hearts appeared to be independent of stimulation rate. The expanded tracings (Fig 7⇓) demonstrate that this effect is not due to fusion of beats.
Effect of Transit Times on Myocyte Contraction
Transplants were performed at two centers—one distant from the laboratory (where transit times were averaged 90 minutes) and one on the same site as the laboratory (where transit times were <5 minutes; indicated with an asterisk in Tables 1⇑ and 2⇑). All biopsies were performed at the center adjacent to the laboratory. We examined the effect of transit times on contraction amplitude and found a small increase in contraction amplitude in myocytes performed at the nearer center compared with those obtained from the remote center in group A patients (Fig 8⇓); this difference was significant (ANCOVA, P<.05). The magnitude of this difference was small compared with the differences observed between failing and nonfailing groups. A comparison between the short transit time transplants and the nonfailing biopsies reveals significant depression of contraction amplitude (ANCOVA, P<.001).
The results of the present study demonstrate that at physiological stimulation frequencies, abnormalities of both contraction and relaxation can be detected at the level of the individual cardiac myocyte in the failing human heart.
The finding of similar degrees of shortening in myocytes from failing and nonfailing hearts at 0.2 Hz and 37°C is in agreement with our previous work,1 demonstrating that the lower temperature (32°C) was not responsible for our failure to detect a difference. The finding of reduced shortening at higher frequencies in isolated cardiac myocytes from failing human hearts demonstrates that myocyte loss12 and abnormalities of the extracellular matrix13 are not solely responsible for the depression of ventricular function in heart failure. The fact that the depressed shortening becomes apparent at higher stimulation rates is in agreement with clinical studies in which the depression of cardiac output in the failing heart occurs at higher heart rates.14 15 Although the majority of the patients in this series had ischemic heart disease as a primary diagnosis, the results obtained in patients with dilated cardiomyopathy were similar, suggesting that it is the presence of long-standing heart failure itself that produces these abnormalities regardless of its underlying cause.
The increased force and amplitude of contraction that occur with increasing stimulation frequency in cardiac muscle fibers was first described by Bowditch11 and has been attributed to increases in the L-type Ca2+ current16 and intracellular [Na+]17 accompanied by reduced sensitivity of the Na+/Ca2+ exchanger.18 Although this effect has been demonstrated to occur both isotonically19 and isometrically20 in isolated guinea pig myocytes, it has not been previously described in isolated human cells. The attenuation or reversal of this effect both in papillary muscle preparations21 22 and clinically14 15 in patients with heart failure has also been described. Although in the majority of reports this negative frequency response occurs in heart failure due to a wide range of causes, some authors have suggested that it is dependent on the underlying cause of heart failure.23 24 The negative force-frequency relation in papillary preparations from failing hearts is potentiated by increased extracellular calcium concentrations23 and reversed by magnesium,25 ouabain,26 isoprenaline,26 and forskolin.27 The underlying mechanisms responsible for these observations remain unclear with experiments from isometric papillary muscles demonstrating both reduced availability of28 and insensitivity to29 intracellular calcium with increasing stimulation frequency in heart failure. In isotonically contracting isolated cardiac myocytes, Beuckelmann et al30 demonstrated a reduction in the calcium transient peak in myocytes from failing hearts at a stimulation rate of 0.5 Hz, whereas in isotonically contracting trabecular preparations Vahl et al31 demonstrated an increased Ca2+ transient in heart failure relative to hearts from control subjects at 1.0 Hz.
Diastolic Shortening in Maximal Ca2+ and 37°C (Group A Patients)
There was no significant difference in the sarcomere lengths of myocytes under basal conditions (0.2 Hz and 1 mmol/L Ca2+), and this is in agreement with previous observations.1 31 In contrast to our previous observations at 32°C,1 the presence of maximally stimulating Ca2+ produced significant shortening of diastolic sarcomere lengths in myocytes from patients with heart failure (Fig 1⇑). Although this observation is consistent with the proposed impairment of diastolic Ca2+ handling in the failing hearts,30 it is possible that impairment of myofilament Ca2+ sensitivity occurring at shorter sarcomere lengths32 contributed to the observed depression of contractility in myocytes from patients with heart failure. The levels of sarcomere shortening that occurred are in excess of those producing sarcomere overlap and contracture. The fact that a comparable depression of shortening in myocytes from failing hearts occurred in group B (with 2 mmol/L Ca2+) suggests that the mechanism of depressed contractility in high Ca2+ is unrelated to small changes in sarcomere length under these experimental conditions.
Diastolic Shortening at 2 mmol/L Ca2+ and 32°C (Group B Patients)
There was no difference in the diastolic sarcomere length with increasing stimulation frequency between myocytes from failing and nonfailing hearts (Tables 7⇑ and 8⇑ and Fig 5⇑). This demonstrates that the failure of the myocytes from impaired ventricles to increase their systolic shortening was not simply due to excessive diastolic contracture.
Characteristics of Contraction and Relaxation
The prolongation of TTP and R50 and R90 in cells from failing hearts at 0.2 Hz is consistent with our previous findings at 32°C.2 In addition, this finding is compatible with descriptions of prolongation of the calcium transient decay occurring in isolated cells from failing hearts30 and in trabecular preparations.31 33 In contrast to this, previous trabecular experiments have failed to demonstrate prolongation of the time to 50% mechanical relaxation at 0.5 Hz,26 1.0 Hz,26 34 or 2.0 Hz26 and in one study, there was a trend toward faster relaxation in muscles from failing hearts.26 That the prolongation of R90 at 0.2 Hz in myocytes from failing hearts becomes less prominent at 1.0 Hz and that this occurs in the absence of diastolic fusion have not been previously described. These findings suggest that some of the well-recognized abnormalities of diastolic function that occur in heart failure35 may originate at the level of the individual cardiac myocyte and that the use of isometric preparations can partially obscure these abnormalities.
Use of Biopsy Samples From Nonfailing Myocardium as Control Samples
There are several potential disadvantages with the use of donor myocardium that has been found to be unsuitable for transplantation as control tissue. First, significant abnormalities of myocardial function can be observed in brain-dead patients,36 and these commonly require inotropic and other support before harvest. Second, the limited availability of such patients is often reflected in the small numbers of control patients in many series. In addition, documentation of the donors’ preharvest cardiovascular status is often poor, and the age range is significantly younger than the population of patients with heart failure. In preliminary experiments using human atrial appendage, we have demonstrated that a threefold improvement in yield can be achieved using vibratome sectioning in place of the traditional razor array with no discernible alteration in myocyte function (unpublished observations). In addition, the results presented here demonstrate similar contractile characteristics at 0.2 Hz to previous experiments using rejected donor hearts.1 Using the technique described here, we have been successful in isolating viable cardiac myocytes from 70% of biopsies and from samples as small as 40 mg. The development of this technique provides a plentiful supply of control tissue and may permit the study of milder forms of ventricular dysfunction in the future.
The use of unloaded preparations clearly does not fully reflect the situation in the intact heart but may represent a useful alternative perspective to traditional isometric preparations, particularly as the heart spends only a small portion of each cycle performing pure isometric work. In addition, recent work describing the attenuation of the Frank-Starling mechanism in papillary preparations from failing hearts37 implies that the lack of an external load would cause us to underestimate the contractile deficit in myocytes from failing hearts compared with myocytes from control subjects with nonfailing hearts.
The second limitation is the use of BDM in the preparation of cells from 5 of the control patients but from none of the cells from failing hearts in group A. However, we do not believe that this significantly affected our results for several reasons. First, we have subsequently isolated cells from two patients from group A (patients 6 and 7) without the use of BDM and all of the patients in group B and from the rejected donor hearts with identical results. Second, the effects of BDM have been shown to be rapidly reversible on washout.38 BDM has been shown not to alter the force-frequency relation in papillary muscle preparations from failing and nonfailing hearts.22 As similar results were obtained with myocytes from nonfailing hearts regardless of whether they were prepared from explanted hearts or from biopsy samples, the differences observed in myocytes from failing hearts cannot be attributed to differences in the preparation techniques. All single myocyte studies have the inherent limitation of the potential for cell selection bias. It should be noted that the myocytes were selected for study at a stimulation rate of 0.2 Hz, where no differences in systolic contractility were apparent. By selecting for study only cells that met our stability criteria, we may have underestimated the true impairment of function among the myocyte population as a whole. Nevertheless, results of the present study demonstrate the profound abnormalities of contraction and relaxation present at higher stimulation rates in myocytes that are apparently morphologically intact. In addition, it should be noted that the depression of contractility is comparable to that seen in papillary and trabecular muscle studies.5 26
This study demonstrates that impaired shortening occurs at the level of the individual ventricular myocyte in human cardiac failure and that myocyte abnormalities can contribute to both systolic and diastolic dysfunction. The production of working myocytes from biopsies of adult human left ventricle has been described.
Selected Abbreviations and Acronyms
|R50||=||time to 50% relaxation|
|R90||=||time to 90% relaxation|
This study was supported by the British Heart Foundation. We are grateful to the Royal Brompton Charitable Trust for the purchase of the Vibratome. Sian Harding is a Wellcome Senior Lecturer. We thank the surgeons and immunologists of Harefield Hospital for their generous help with the samples of explanted hearts, and we express our gratitude to the patients who kindly donated myocardial biopsies.
- Received January 10, 1995.
- Revision received May 29, 1995.
- Accepted June 3, 1995.
- Copyright © 1995 by American Heart Association
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