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J Thorac Cardiovasc Surg 2001;121:902-908
© 2001 The American Association for Thoracic Surgery
Surgery for Acquired Cardiovascular Disease |
From the Departments of Medicinea and Surgery,b Columbia University, New York, NY.
Received for publication March 15, 2000. Revisions requested April 12, 2000; revisions received Oct 26, 2000. Accepted for publication Oct 31, 2000. Address for reprints: John D. Madigan, c/o Daniel Burkhoff, Columbia University, Department of Medicine, Black Building 812, 650 West 168th St, New York, NY 10032 (E-mail: jdm50{at}columbia.edu).
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Abstract |
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Introduction |
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1 regression of LV myocyte hypertrophy, 2 alterations in LV myocardial collagen content, and normalized expression of genes involved with excitation-contraction coupling. 3 Collectively, these changes have been referred to as reverse remodeling 1 and, in some circumstances, lead to improved intrinsic myocyte strength. 3,4 In a small number of cases, the reverse remodeling leads to recovery of chamber strength of sufficient magnitude to permit LVAD explantation without the need for transplantation. 5-7 Although clinical outcomes after LVAD explant have not been uniformly good 6 and LVAD explant is not routine in the United States, understanding the process of reverse remodeling is important. Such understanding will lead to new insights into fundamental mechanisms underlying heart failure, the extent to which pathophysiologic processes can be reversed, and the potential role of LVAD therapy as an adjunct to other therapies aimed at curing heart failure. More recent evidence suggesting that long-term outcome after LVAD explantation could be improved with appropriate patient selection and weaning protocols 7 make improved understanding of the process even more important. Current understanding of LVAD-induced reverse remodeling is rudimentary. One fundamental unexplored aspect of this multifaceted process is the time course with which it occurs. The objective of this study was therefore to define the time course of structural, cellular, interstitial, and molecular reverse remodeling of the left ventricle during LVAD support.
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Methods |
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All hearts were perfused with cold, hypocalcemic, hyperkalemic cardioplegic solution at explantation. The passive LV pressure-volume relationship of each arrested heart was measured as described previously. 3 In brief, the aortic root and, in the case of the LVAD-supported hearts, the LVAD inflow cannula were clamp occluded. A metal adapter was attached to the mitral anulus, and a compliant water-filled latex balloon was placed within the LV chamber. Pressure within the balloon was measured with a high-fidelity micromanometer as volume was progressively increased. The pressure was then plotted as a function of volume at each step, resulting in a passive LV pressure-volume relationship that is similar to the end-diastolic pressure-volume relationship of a contracting heart. Chamber size was indexed by the volume at which pressure within the ventricle reached 30 mm Hg (LVV30).
Myocyte diameter and myocardial collagen content
Tissue samples were obtained from the LV free wall, fixed in 10% buffered formalin, embedded in paraffin, and mounted on glass slides. Samples were then prepared with Masson trichrome stain. Images were viewed on a Nikon microscope (Nikon Corporation, Tokyo, Japan) with an MTI 3CCD digital camera (Dage-MTI, Inc, Michigan City, Ind) at 20x magnification to assess myocyte diameter and at 4x magnification to assess myocardial collagen content. Digitally acquired images were analyzed with Image Pro Plus V3.0 software (Media Cybernetics, LP, Silver Spring, Md) by an examiner blinded to whether the heart was normal, failing, or LVAD supported. Two orthogonal diameters were obtained per myocyte and then averaged. Only sections containing fibers cut in cross-section were analyzed. The diameters of 50 myocytes per patient were measured and then averaged. Myocardial collagen content was assessed by using a green software filter. On each section, a density range was set to resemble the collagen content. The number of pixels included in this range was divided by the total number of pixels on the fields. Ten fields at 4x magnification per patient were analyzed, and results were then averaged.
Northern blot analysis
Tissue samples were obtained from the LV free wall and snap-frozen in liquid nitrogen for RNA extraction. Total RNA was extracted from the myocardial tissue samples with guanidinium thiocyanate followed by centrifugation in cesium chloride solutions. Aliquots (25 µg) of total RNA quantified by absorbance at 260 nm were then separated by using electrophoresis in 1% agarose/15% formaldehyde gels in 1x 3-(N-morpholino) propanesulfonic acid buffer (Fisher Scientific, Hampton, NJ). After intensive capillary transfer to Nirto Pure nitrocellulose membranes (MSI, Westboro, Mass) in 20x standard saline citrate (SSC; 1x SSC equals 3 mol/L sodium chloride and 0.15 mol/L sodium citrate, pH 7.0), the RNA was fixed by baking in a vacuum oven at 80°C for 1 hour and prehybridized at 42°C for more than 3 hours in a solution of 50% formamide, 4x SSC, 1x Denhardt solution, 0.1% sodium dodecylsulfate (SDS), 1 mmol/L ethylenediaminetetraacetic acid, and 0.125 mg/mL denatured salmon testis DNA. The blots were hybridized overnight with cDNA for rat sarcoplasmic endoreticular calcium adenosine triphosphatase 2 (SERCA2; 1.181-kb EcoRI fragment, a generous gift from Dr Gerd Hasenfuss) and human reduced glyceraldehyde-phosphate dehydrogenase (GAPDH; 1.3-kb PstI fragment). All cDNA probes were labeled with a phosphorus 32–labeled deoxycytidine triphosphate (3000 Ci/mmol, Amersham, Arlington Heights, Ill) to a specific activity of 1 x 106 cpm/µg by using a multiprimer DNA-labeling system (Amersham). The blots were washed twice in 2x SSC at room temperature for 15 minutes and 3 times in 0.1x SSC/0.1% SDS at 68°C for 15 minutes and exposed to X-OMAT AR film (Eastman Kodak Company, Rochester, NY) at –70°C with an intensifying screen for 48 to 72 hours. Autoradiograms were then analyzed by means of laser densitometry (Molecular Dynamics Inc, Sunnyvale, Calif) in the linear response range of the x-ray films by using GAPDH as an internal standard. The relative SERCA2a expression was quantified as the ratio of SERCA2a band intensity to GAPDH band intensity. To be able to pool results from different gels, all values were further standardized to the average intensities of the same 3 normal samples that were included on every blot. Of the major proteins involved in calcium cycling that we have examined previously (which have included the ryanodine receptor and sodium-calcium exchanger in addition to SERCA2a), it is our experience that SERCA2a mRNA levels are the highest under normal conditions and thus provide the greatest reliability for detecting changes in expression. 3
Data analysis
Data related to each parameter examined were graphed as a function of duration of LVAD support and fit to an exponential function as follows:
y = yo + 
(1-e–t/
)
where y is the parameter of interest, yo is the extrapolated value to 0 days of support, t is the duration of LVAD support, is the maximum magnitude of the change, and is the time constant for change. Fitting was done with a nonlinear Levenberg-Marquard algorithm. To determine whether these relationships where statistically significant, they were linearized by means of a standard logarithmic transformation. Linear regression analysis was then applied, which provided a P value for the relationship.
As detailed further in the "Results" section, patients were divided into 4 groups on the basis of duration of support for the purpose of performing statistical comparisons. The normal group (n = 5) comprised hearts from normal donors. The non-LVAD group (n = 19) comprised hearts from patients in end-stage CHF who did not receive LVAD support before transplantation. The LVAD0-40 group (n = 6) comprised hearts from patients with end-stage CHF supported for less than 40 days before transplantation. The LVAD40+ group (n = 19) comprised hearts from patients with end-stage CHF supported for more than 40 days before transplantation. The choice of 40 days as the cutoff point for this grouping was based on the longest time constant of change determined from the nonlinear analysis. Differences between the groups were analyzed by means of 1-factor analysis of variance with a Tukey-Kramer post hoc multiple comparisons test. All values are presented as means and SDs.
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Results |
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) revealed a time constant of approximately 30 days. Statistical comparisons revealed no difference between LVV30 values of the non-LVAD and LVAD0-40 groups (P = .14) and statistically significant differences between the non-LVAD and LVAD40+ groups (P < .001) and LVAD0-40 and LVAD40+ groups (P < .001) and between the control group and each of the other 3 groups (P = .015). Thus although substantial chamber reverse remodeling had occurred with LVAD durations greater than 40 days, LVV30 on average was still greater than that of the normal hearts. These differences are shown more clearly in the mean passive pressure-volume curves for the normal, non-LVAD, LVAD0-40, and LVAD40+ hearts shown inFig 1, A.
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, B). The time constant of change of myocyte diameter was approximately 24 days. There was a significant difference between the non-LVAD and normal groups (P < .001), and no statistically significant difference between the non-LVAD and LVAD0-40 groups (P = .18). The LVAD40+ group exhibited significantly smaller diameters when compared with the non-LVAD group (P = .002), although the values were still significantly larger than those in the normal hearts (P = .01). Examples of histologic samples are shown inFig 2, A (normal), B (LVAD0-40), and C (LVAD40+).
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, B (format similar to that ofFig 1, B). As in previous studies, SERCA2a expression was significantly decreased in the non-LVAD patients undergoing CHF and in the LVAD0-40 group compared with the normal group (P < .001 and P = .005, respectively), and there was no statistically significant difference between the non-LVAD and LVAD0-40 groups (P = .18). The LVAD40+ group, however, exhibited values significantly higher than those of the non-LVAD group (P < .001) and reached values that were not different than those of the normal hearts (P = .30). The time constant of change of SERCA2a determined by the nonlinear curve fitting was approximately 15 days, which is shorter than those of the other parameters examined.
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, B). In contrast to the other parameters studied, there was no statistically significant difference between the non-LVAD and normal groups (P = .24). There was also no statistically significant difference between the non-LVAD and LVAD0-40 groups (P = .10). However, the LVAD40+ group exhibited values significantly higher than those of the non-LVAD group (P < .001). The blinded nature of this analysis and the attempt to avoid areas of massively fibrotic areas should be reiterated. The time constant of change of interstitial collagen was about 40 days, which is slightly longer than the other parameters. Subgroup analysis showed that this finding was independent of the cause of CHF (ischemic cardiomyopathy vs dilated idiopathic cardiomyopathy). Thus LVAD support may lead to an increase in relative myocardial collagen content. Examples of histologic sample are shown inFig 4, A (normal), B (LVAD0-40), and C (LVAD40+).
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Discussion |
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Relative collagen content increased during LVAD support. It is likely that this is purely a result of the decrease in myocyte mass that occurs during LVAD support rather than increased deposition of collagen. It is clear that a decrease in myocyte mass with a fixed absolute amount of collagen would result in an increase by the same percentage of myocardium occupied by collagen. By using a simple model of myocardium composed only of myocytes and collagen, it can be shown quantitatively that a reduction of myocyte diameter from 35 to 25 µm would be expected to increase relative collagen content from the measured starting value of 10% to a predicted value of 14% if the absolute amount of collagen remained constant. This is very close to the measured 15% collagen content determined for the LVAD40+ group by means of blinded analysis(Fig 4, D). Additionally, the half-life of collagen turnover is between 80 and 120 days, and therefore the patients with LVADs included in the present study did not have durations of support long enough to allow for significant alterations in collagen deposition or resorption. Significantly longer durations of support would be required to determine whether LVAD support influences absolute collagen content.
The precise mechanisms underlying the various aspects of reverse remodeling remain to be determined. The mechanisms underlying hypertrophy and chamber remodeling in response to pressure and volume loading of the myocardium involves intricately orchestrated upregulation and downregulation of a multitude of intracellular signaling cascades. 8 Although investigated for over 20 years, these mechanisms are still not fully understood. It has been our working hypothesis that reverse remodeling involves the same mechanisms working in the opposite direction. In fact, it has been questioned whether reverse remodeling is simply a reflection of myocyte atrophy. The finding that, on average, myocyte diameters in the LVAD40+ group were still larger than normal and that the process of cellular size reduction appears to be completed by approximately 40 days despite continued off-loading of the muscle implies that there is a limit to how much atrophy the once diseased and hypertrophied myocyte can undergo.
Recent evidence suggests that in some patients, LVAD support may lead to improvement of global pump function of sufficient magnitude to permit explantation of the device without subsequent transplantation. 5 This has led to the concept of using LVADs as a bridge to recovery. The potential of this possibility is strengthened by data demonstrating normalization of LV chamber geometry, 1 regression of LV myocyte hypertrophy, 2 increased myocyte contractile strength, enhanced inotropic response to adrenergic stimulation, and improved cytosolic calcium transients. 4 However, in a recent article from our institution, there was a low incidence of myocardial recovery during LVAD support, as assessed by exercise testing with device output turned down, and the outcome of a small group of patients that underwent explantation was not uniformly good. 6 Accordingly, weaning from LVAD support is not currently considered the standard of care, although more recent data suggest that outcome after LVAD explant may be improved. 7 The goal of using LVADs as a bridge to recovery is a worthy pursuit because of the severe imbalance between the number of patients requiring transplant and the number of available donor hearts. Better understanding of the process of reverse remodeling will aid the development of adjunctive therapies, better patient selection criteria, and perhaps optimum LVAD use protocols to improve patient outcome after LVAD explantation. The data of the present study indicate that, as used currently, maximum benefits of LVADs in terms of reverse remodeling are observed after about 40 days, and little change is seen after this time point. In the short term a small increase in relative interstitial fibrosis may occur, not because of an increase in collagen deposition but because of the decrease in myocyte diameter with no significant change in total collagen content.
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