Reversal of Myocyte Hypertrophy by Ventricular Unloading: Cardiac Improvement Without Adrenergic Receptor Upregulation and Relocalization
Abstract
In previous studies, we found that the improved contractile ability of cardiac myocytes from patients who have had left ventricular assist device (LVAD) support was due to a number of beneficial changes, most notably in calcium handling (increased sarcoplasmic reticulum calcium binding and uptake), improved integrity of cell membranes due to phospholipid reconstruction (reduced lysophospholipid content), and an upregulation of adrenoreceptors (increased adrenoreceptor numbers). However, in the case presented here, there was no increase in adrenoreceptor number, which is something that we usually find in core tissue at the time of LVAD removal or organ transplantation; also, there was no homogeneous postassist device receptor distribution. However, the patient was well maintained for 10 months following LVAD implantation, until a donor organ was available, regardless of the lack of adrenoreceptor improvement.
We conclude from these studies that cardiac recovery is the result of the initiation of multiple repair mechanisms, and that the lack of expected changes, in this case increased adrenoreceptors, is not always an accurate indicator of anticipated outcome. We suggest that interventions and strategies have to consider multiple, beneficial changes due to unloading and target a number of biochemical and structural areas to produce improvement, even if not all of these improvements occur.
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Introduction
We have published findings on the recovery of heart failure patients following left ventricular unloading with the implantation of a left ventricular assist device (LVAD).[1,2] Mechanical indexes show that contractile function, flow parameters, and ejection fraction (EF) are all improved following LVAD surgery, to the extent that the initial drive to develop assist devices as a bridge to transplantation has been replaced by the possibility of LVAD as a bridge to recovery.
Initial studies revealed that the heart recovered and that there was cellular and subcellular repair. Calcium handling mechanisms and pathways were rectified; fibrosis was reduced; myocyte hypertrophy was reversed; and leaky membranes were patched. Overall, biochemical and pathologic studies on tissue from patients at the time of LVAD removal gave us many more positives than we could have envisioned.
In our collaborations, we have identified a trend that appears to dictate a favorable outcome for LVAD recipients,[3,4] a trend that has been noted by other laboratories and research groups.[5–7] Adrenoreceptors downregulate when the heart becomes stressed and unable to perform efficiently. The hypothesis is that the adrenoreceptor-mediated calcium signaling pathways are “switched off” to avert calcium overload and cell death. However, the responsiveness of beta receptors is returned to normal levels following mechanical unloading, and we have found that alpha-adrenergic receptors, which have been overlooked players in heart failure, also follow this reversible downregulation phenomenon.[4]
We therefore used blind correlations to attempt to predict patient outcome in regard to whether continued LVAD support was implemented or if the patient was transplanted, which depended on our results from receptor distributions and numbers in core biopsies. Our predictions of 32 patients were mostly accurate (unpublished data). Therefore, it was somewhat surprising to us that in the patient who we detail here, who was supported by a HeartMate pump (Thoratec, Pleasanton, California), there seemed to be no changes in receptor number (upregulation) and no redistribution to the myocytes following LVAD implantation. However, the patient fared well on the LVAD to the extent that mechanical indexes and the EF improved (EF < 20% pre-LVAD, 20% post LVAD; LVEF 0.206 pre-LVAD, 0.270 post LVAD). Both the pre- and post-LVAD samples were imaged extensively, acquiring many areas of the myocardium.
Reversal of myocyte hypertrophy is common after ventricular unloading, but in this individual the reversal was to such an extent that the other, usual improvements might have been “unnecessary” for a positive, reparative response.
Methods
Tissue Preparation and Staining
After biopsy, the fresh cardiac tissue samples were embedded in 10.24% polyvinyl alcohol, 4.2% polyethylene glycol, and 85.5% sucrose (Tissue-Tek OCT Compound, Torrance, California) and immediately frozen on dry ice. When the samples reached 4°C, the blocks were sectioned (thickness, 10 ± 3 mcm) with a Reichert HistoSTAT cryotome, which was attached to poly-L-lysine-coated (Sigma) glass coverslips and placed in 3.7% paraformaldehyde for 5 minutes at room temperature. Samples were viewed with an Applied Precision DeltaVision scanning fluorescence microscope (Issaquah, Washington) fitted with an Olympus IX70 microscope (Melville, New York) and deconvolution capabilities. Sections were stained with the appropriate fluorescence receptor probes (5 nmol/L for 30 minutes at 37°C) and placed on a glass slide with 1 drop of Elvanol (DuPont Antifade, Wilmington, Delaware). For localizing cell nuclei, a DAPI probe (4′,6′-diamidino-2-phenylindole, 0.1 g/mL; Molecular Probes, Inc., Eugene, Oregon) was used, and to identify alpha-1 adrenoreceptors (alpha1ARs) a BODIPY 558/568-tagged prazosin probe (Molecular Probes, Inc.) was used. We also used primary antibodies against cardiac myosin, with BODIPY green-labeled and Texas Red-labeled secondary antibodies (Molecular Probes, Inc.) to visualize the contractile protein. A Kd of 0.13 nmol/L for prazosin was obtained. Nonspecific binding was monitored by preincubating tissue with cold prazosin prior to the tagged probe, and by incubating tissue prior to labeling with other alpha-1 agonists and antagonists (Tocris-Cookson, St. Louis, Missouri; RS 17053 an alpha-1A antagonist; clonidine, an alpha-2 agonist and M-6434, an alpha-1 agonist) to ensure that specificity of the fluorescent probe was high. Beta-adrenoreceptor typing was done with CGHP-12177, a generic tag, following preincubation with the compounds BRL 37344, which is specific for beta3 receptors; CGP 207 (beta1); and 1C1 118,551 (beta2). These probes have been used in previously published work.[8–10]
Image Acquisition
Images (thickness, 0.25 mcm) were acquired in a complete pass from bottom to top of the tissue, and then subjected to deconvolution (5 iterations), stacking, and volume rendition with Imaris software (Bitplane AG, Zurich, Switzerland). Stereology consisted of counting 3 distinct areas of fluorescence in 3 images from the same tissue sample (9 counts/sample) to reduce potential errors. Areas of interest were captured as red-green-blue (RGB) files (90 × 90 microns); the gain was set to accentuate individual points of intense fluorescence and to maximize clarity. Mean values for receptor density were determined as the number of pixels in a 60 × 60-mcm area. Measurements were performed and cross-checked with both a Corel and a SigmaScan program (Corel Corp., Ottawa, Ontario, Canada, and SPSS Inc., Plover, Wisconsin, respectively). We measured the cross-sectional dimensions (n = minimum of 30 fibers) in tissues before and after LVAD insertion.
Cell-Type Recognition
To determine the cell types in our sections (predominantly fibroblasts, endothelial cells, and myocytes), we used a number of probes: DAPI (0.1 g/mL; Molecular Probes, Inc.) to identify nuclei, a primary antibody (SMA Monoclonal, Sigma, diluted 1:100) followed by a secondary AlexaFluor 647 antibody (Molecular Probes, Inc., goat anti-mouse, diluted 1:500) to probe for smooth-muscle actin, and BODIPY and Texas Red-tagged phallacidin (1:100) against cardiac actin (Molecular Probes, Inc., diluted 1:100).
Results
Cross-sectional measurements of fiber diameters were post LVAD 4.98 ± 2.74 vs pre-LVAD 10.74 ± 1.10 microns (P < .05; ± SEM). These contrasting widths are shown in the pairs that are presented in Figure 1 (yellow brackets), in which A and B are cross sections, C and D are longitudinal sections, and Panels A and C are post-LVAD samples. Note the tight fiber profile in Panel A compared with the ragged-looking fibers in Panel B. Also of note, the fibers in C have no intercontractile fiber gapping and demonstrate obvious fiber branching and intercalated discs. We believed that a surprising observation was the equivalent amount of receptors in both the pre- and post-LVAD samples (Panels A and B show beta2; Panels C and D show alpha1D receptors) when we measured distributions in 15 areas of each tissue (7037 vs 3519, A vs B; 3861 vs 2227, C vs D: overall 6366 ± 4233 for post LVAD, 4888 ± 2976 for pre-LVAD).
Four images from the reported patient demonstrate cross sections (A and B) and longitudinal sections (C and D) for post-left ventricular assist device (LVAD) (A and C) and pre-LVAD (B and D) samples. Note the width of the myofibrils (green fluorescent tag, indicated by the yellow brackets) and the clumped appearance of these receptors (Texas Red tag, Panel C), even after unloading. Image acquisition is as described under the “Methods” section, and each panel is 90 × 90 microns.
Comparing the images from Figure 1 with those in Figure 2 from another subject in our study who received LVAD support, it is obvious that in normal explant (post-LVAD) tissue from patients who demonstrate adrenoreceptor increases (first panel, Figure 2), the cellular integrity improved; the number of adrenoreceptors (red) increased; however, more importantly, the receptors are homogeneously spread throughout the tissue when compared with the implant (pre-LVAD insertion) tissue and the images from the “nonresponsive” patient in Figure 1. The adrenoreceptor upregulation and relocalization was achieved, as seen in Figure 2, as is usually found after LVAD support.
Two images of explant vs implant tissue, demonstrating the copious number and distribution of adrenoreceptors after ventricular unloading (first panel), from a second patient in this study, compared with the sparse number of adrenoreceptors before unloading and the disjointed myofibers before unloading. Images are stained for actin and beta adrenoreceptors in this example. Image size is 96 × 96 microns, and acquisitions were made as described under “Methods.”
Discussion
Our hypothesis was that to some degree of accuracy we could make an educated guess as to a patient's outcome from measurements of adrenoreceptors in tissue that has been removed at the time of LVAD implantation, and observe the amount of recovery/repair in the organ at the time of transplantation or autopsy. This proved to be the case in most instances. However, in this patient there was not a significant increase in adrenoreceptor number or change in adrenoreceptor localization, something that we would have expected after mechanical ventricular unloading, although there was a dramatic improvement in tissue integrity. These findings point to the multiple factors that are involved in the repair and recovery of the myocardium and add weight to the strategy that was proposed by Hon and Yacoub[5] in 2003, and to other clinical efforts in the ensuing years, that targeting of beta2 adrenoreceptors should be a major consideration in the treatment of heart failure patients (eg, McBride and White[11]), especially given the benefits of mechanical unloading and that improvement isn't solely limited to adrenoreceptor upregulation.
The findings also direct us to consider the role of alpha adrenoreceptors in cardiac recovery while under the influence of an assist device, particularly when these receptors have been implicated in depressed cardiac function,[10] and to recognize that not all of the expected changes have to occur for a patient to be successfully supported by LVAD until organ transplantation.
Contributor Information
Pippa M. Schnee, Department of Pathology, University of Texas Medical School at Houston.
Naeema Shah, Texas Heart Institute, Houston, Texas.
Marianne Bergheim, Texas Heart Institute, Houston, Texas.
Brian J. Poindexter, University of Texas Medical School at Houston.
L. Max Buja, University of Texas Medical School at Houston; Cardiovascular Pathology, Texas Heart Institute, Houston, Texas.
Timothy J. Myers, Cardiovascular Surgery and Transplant Research Pathology, Texas Heart Institute, Houston, Texas.
Branislav Radovancevic, Cardiovascular Surgery and Transplant Research, Texas Heart Institute, Houston, Texas; University of Texas Health Science Center at Houston.
O. Howard Frazier, Texas Heart Institute, Houston, Texas.
Roger J. Bick, Department of Pathology, University of Texas Medical School at Houston. Email: ude.cmt.htu@kcib.j.regor.