Surgery for Acquired Cardiovascular Disease

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1 Suzuki et al Surgery for Acquired Cardiovascular Disease The reduction of hemodynamic loading assists self-regeneration of the injured heart by increasing cell proliferation, inhibiting cell apoptosis, and inducing stem-cell recruitment Ryo Suzuki, MD, Tao-Sheng Li, MD, PhD, Akihito Mikamo, MD, PhD, Masaya Takahashi, MD, Mako Ohshima, MS, Masayuki Kubo, PhD, Hiroshi Ito, MD, PhD, and Kimikazu Hamano, MD, PhD Objectives: Mitotic cardiomyocytes and cardiac stem cells have been identified recently in adult hearts, and both have been found to be increased in acute infarcted myocardium. Although these findings suggest potential self-repair of the heart after injury, obvious self-regeneration of the injured heart has never been observed clinically. We hypothesized that hemodynamic loading impairs myocardial repair. Dr Suzuki, Prof Hamano, and Dr Li (left to right) From the Department of Surgery and Clinical Science, Division of Cardiac Surgery, Yamaguchi University Graduate School of Medicine, Ube, Japan. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture and by the JSPS Fujita Memorial Fund for Medical Research. Received for publication Sept 25, 2006; revisions received Dec 8, 2006; accepted for publication Dec 13, Address for reprints: Tao-Sheng Li, MD, PhD, Department of Surgery and Clinical Science, Division of Cardiac Surgery, Yamaguchi University Graduate School of Medicine, Minami-Kogushi, Ube, Yamaguchi , Japan ( litaoshe@yamaguchi-u.ac.jp). J Thorac Cardiovasc Surg 2007;133: /$32.00 Copyright 2007 by The American Association for Thoracic Surgery doi: /j.jtcvs Methods: Myocardial infarction was induced in C57BL/6 mice by ligating the left anterior descending artery. After 60 minutes, either the infarcted heart was transplanted heterotopically into a healthy recipient C57BL/6 mouse to remove the ventricular hemodynamic loading (unloading group) or it was left as an infarcted heart under normal hemodynamic loading conditions in the same mouse (loading group). The infarcted hearts were dissected for histologic analysis after 3, 7, 14, and 28 days. Results: Histologic analysis showed that the wall thickness of the infarcted left ventricle was significantly greater and the area of infarction was significantly smaller in the unloading group than in the loading group. Immunostaining analysis revealed significantly more Ki-67-positive cells and significantly fewer apoptotic cells in the infarcted myocardium in the unloading group than in the loading group. There were also significantly more c-kit- and Sca-1-positive stem cells in the infarcted myocardium in the unloading group than in the loading group. Conclusion: Our findings suggest that hemodynamic unloading assists selfregeneration of the injured heart by increasing cell proliferation, inhibiting cell apoptosis, and inducing stem-cell recruitment. Unlike the skin, liver, muscle, and other organs, it is still thought that the heart cannot regenerate because of the loss of proliferative potential of adult cardiomyocytes and the lack of cardiac stem cells in the adult mammalian heart. 1 However, recent investigations have provided evidence that adult cardiomyocytes do retain limited cell cycle activityand that there are in fact cardiac stem 2,3 4-7 cells in the adult heart. Furthermore, an increase in either mitotic cardiomyocytes 2,3 or cardiac stem cells has been identified in failing and infarcted hearts. These findings suggest that the injured heart may potentially have regenerative function; however, obvious myocardial regeneration has never been observed clinically after heart injury. The process of repairing the damaged heart is thought to be related to the balance between regeneration and loss of myocytes. Although the increased number of mitotic cardiomyocytes and cardiac stem cells in the infarcted heart will accelerate the regeneration of new myocardium, an excessive loss of cardiomyocytes may also The Journal of Thoracic and Cardiovascular Surgery Volume 133, Number

2 Surgery for Acquired Cardiovascular Disease Suzuki et al Abbreviations and Acronyms DAPI 4=, 6-diamidino-2-phenylindole LV left ventricular LVAD left ventricular assist device PE phycoerythrin SDF-1 stromal cell derived factor 1 TUNEL terminal deoxynucleotidyl transferasemediated dutp nick end-labeling be induced by the ventricular mechanical stresses and severe milieu in the infarcted heart. Because the frequency 8,9 of mitotic cardiomyocytes in humans is very low (about 0.015% in the failing heart and 0.08% in the acute infarcted heart), 2,3 a negative balance between regeneration and loss of myocytes might provide a reasonable explanation of why self-repair of the damaged heart does not occur clinically. Interestingly, substantial recovery of cardiac function has been achieved by the implantation of a left ventricular assist device (LVAD) in some patients with end-stage heart failure, and the device has even been explanted successfully in some of these patients. A beneficial effect of LVAD support after coronary artery bypass grafting in patients 13 with acute coronary occlusions has also been reported. Although the precise mechanism of self-repair of the injured heart under LVAD support is unclear, we speculate that the reduction in ventricular mechanical stress achieved by LVAD support inhibits the loss of myocytes, resulting in a positive balance between the regeneration and loss of myocytes. In this study, we placed the left ventricle of infarcted hearts under hemodynamic unloading conditions by heterotopic transplantation and then investigated the role and relative mechanisms of hemodynamic loading in myocardial repair. Materials and Methods Animals Male, 10-week-old C57BL/6 mice (16 18 g) from Japan SLC (Shizuoka, Japan) were used in these experiments, which were approved by the Institutional Animal Care and Use Committee of Yamaguchi University. The animals were bred in clean conditions and allowed free access to food and water in a temperaturecontrolled environment with a 12-hour light/12-hour dark cycle. This investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No , revised 1996). Myocardial Infarction Model and Experimental Protocol A myocardial infarction model was established in C57BL/6 mice 14 as described previously. Briefly, after inducing general anesthe - sia with pentobarbital and performing tracheal intubation with a 20-gauge intravenous catheter, the mice were artificially ventilated with room air at 80 breaths per minute. We performed a left thoracotomy through the fourth intercostal space and ligated the left anterior descending artery completely with 8 0 polypropylene under direct vision. To estimate how the left ventricular (LV) hemodynamic loading effects myocardial regeneration, the infarcted hearts were randomly subjected to an unloading condition or a normal loading condition 60 minutes after ligation of the left anterior descending artery. The normal LV hemodynamic loading condition (loading group, n 29) was created simply by leaving the infarcted heart in the same mouse and performing a sham laparotomy. The LV hemodynamic unloading condition was created by heterotopic transplantation of the infarcted heart into another healthy C57BL/6 mouse (unloading group, n 27). Briefly, 60 minutes after left anterior descending artery ligation, the infarcted heart was flushed with 1 ml of cold cardioplegic solution (Na 85.3 mmol/l, K 25.0 mmol/l, Cl 85.5 mmol/l, Mg 10.0 mmol/l, and glucose 25 g/l; ph 7.38) via the inferior vena cava and harvested routinely as a donor heart. Then, the infarcted donor heart was transplanted immediately into the abdomen of another normal C57BL/6 mouse, with anastomosis of the donor ascending aorta to the recipient abdominal aorta and of the donor pulmonary artery to the recipient 15 inferior vena cava, as described previously. The infarcted heart resumed vigorous contraction within 3 minutes of reperfusion. Similar to LVAD support, this donor heart provided hemodynamic unloading to the infarcted left ventricle but coronary perfusion was sustained. Sample Collection and Morphologic Observation All the mice were killed and the infarcted hearts were harvested 3, 7, 14, and 28 days after the operation (n 5 9 at each time point for both groups). The infarcted hearts were flushed thoroughly with saline solution. After excising the atrium and other tissues, we recorded the ventricular weight of each heart. The ventricle was cut into 5 pieces for macromorphologic observation of cross sections (about 1.5 mm thick). Samples were embedded in optimal cutting temperature compound and snap-frozen in liquid nitrogen. Histologic analysis was done on 5- m-thick frozen sections. Histologic Analysis Hematoxylin eosin staining and Azan staining was done to estimate the LV wall thickness and infarction area, respectively. Using Image-Pro image analysis software (version 5.1.2, Media Cybernetics Inc, Carlsbad, Calif), the LV wall thickness and fibrotic area in each digital picture were measured quantitatively by a single observer blind to the treatment regimen. The mean wall thickness was measured from 3 equidistant points, and the infarction area was calculated as the area stained blue. Measurements were done in at least 5 separated sections of each heart, and the averages of each heart were used for statistical analysis. Measurement of the Proliferation and Apoptosis of Cells in the Infarcted Hearts The cell proliferation was identified by immunostaining with phycoerythrin (PE)-labeled Goat anti-mouse Ki-67 antibody (1:20 dilation, Santa Cruz Biotechnology, Inc, Santa Cruz, Calif). The apoptosis of cells was detected by a terminal deoxynucleotidyl transferase-mediated dutp nick end-labeling (TUNEL) method, 1052 The Journal of Thoracic and Cardiovascular Surgery April 2007

3 Suzuki et al Surgery for Acquired Cardiovascular Disease Figure 1. Morphologic and histologic findings of self-repair of the infarcted heart under loading and unloading conditions. A, The anterior wall of the infarcted left ventricle was replaced completely by thin scar tissue (arrow) in the loading heart (left), whereas the left ventricle anterior wall appeared to be thick with less scar tissue (arrowhead) in the unloading heart (right) 28 days after infarction. B, The ventricular weight decreased gradually in the unloading group but was increased 28 days after infarction in the loading group. Closed bar, loading group; open bar, unloading group; *P <.05, **P <.01. C, Hematoxylin eosin staining showed that the infarcted myocardium was replaced completely by fibrotic tissue in the loading heart 28 days after infarction, whereas layers of cardiomyocytes (arrow) were observed distinctly in the endocardium of the infarcted left ventricle in the unloading heart. D, Quantitative analysis showed that the wall thickness of the infarcted left ventricle was significantly less in the loading group than in the unloading group 28 days after infarction. E, Azan staining showed a larger fibrotic area (blue) in the loading heart than in the unloading heart. F, Quantitative analysis showed that the area of infarction was significantly greater in the loading group than in the unloading group 28 days after infarction. using Apoptosis Detection Kits (R&D System Inc, Minneapolis, Minn). Sections were also stained with DAPI (4=, 6-diamidino-2- phenylindole) to visualize the nuclei. The number of positive cells was counted under 400-fold magnification by a single observer blind to the treatment regimen, and 20 different fields on 3 independent slides from different cross sections were randomly selected for each heart. We calculated the mean number of positively stained cells per field in the infarcted myocardium for statistical analysis. Detection of Cardiac Stem Cells and Stromal Cell-derived Factor Expression in the Infarcted Hearts To measure the number of stem cells in the infarcted hearts, 5- m-thick frozen sections were stained with PE-labeled rat antimouse c-kit antibody (1:20 dilation, ebioscience, San Diego, Calif) and rabbit anti-mouse Sca-1 antibody (1:20 dilation, R&D Systems). The number of positive cells was counted under 400- fold magnification by a single observer blind to the treatment regimen, and 20 different fields on 3 independent slides from different cross sections were randomly selected for each heart. We calculated the mean number of positively stained cells per field in the infarcted myocardium for statistical analysis. We also examined the expression of stromal cell-derived factor 1 (SDF-1), one of the most important factors for mediating stem cells recruitment and homing. Frozen sections were stained with rat polyclonal antibody against SDF-1 then a universal LSAB2 alkaline phosphatase kit and fuchsin (Dako) for color reaction were used to visualize the immune reaction. Statistical Analysis Results of quantitative studies are expressed as means SD. Statistical comparisons between groups were performed by the The Journal of Thoracic and Cardiovascular Surgery Volume 133, Number

4 Surgery for Acquired Cardiovascular Disease Suzuki et al Figure 2. The proliferation of cells in the injured heart. A, Representative photograph of the proliferating cells. The proliferation of cells was identified by immunostaining with the nuclear antigen of Ki-67 (red, left), and nuclei were stained by DAPI (blue, middle). Scale bars: 20 m. B, Quantitative counting of Ki-67-positive cells with a myocyte-specific morphologic structure revealed significantly fewer proliferating cells in the loading group than in the unloading group 7 days after infarction. unpaired Student t test using StatView software (version 5.0). Values of P.05 were considered significant. Results Myocardial Repair of the Infarcted Hearts Repair of the infarcted hearts was evaluated 28 days after left anterior descending artery ligation. The LV wall was replaced by a thin layer of white scar tissue in the normal LV hemodynamic loading heart (Figure 1, A and C), whereas the LV wall was obviously thicker (Figure A), 1, and a band of surviving myocardium was seen clearly in the unloading heart (Figure 1, C). The ventricular weight decreased gradually in the unloading heart (Figure B), 1, probably as a result of unloading-related cardiac atrophy. The ventricular weight remained stable in the loading heart during the first 2 weeks but was increased at 28 days (Figure Proliferation and Apoptosis of Cells in the Infarcted Hearts We measured the proliferation and apoptosis of cells by immunostaining analysis 3 and 7 days after infarction. As we did not perform double staining, we counted only Ki- 67-positive cells and TUNEL-positive apoptotic cells with a myocyte-specific morphologic structure microscopically. We observed Ki-67-positive cells and TUNEL-positive apoptotic cells mainly within the border area and the infarction area (Figure 2, A and Figure 3, A). Quantitative analysis revealed significantly more Ki-67-positive cells in the infarcted myocardium in the unloading group than in the loading group on day 7 P (.01, Figure 2, B), but there were no significant differences between the groups on day 3 (P.12, Figure 2, B). Conversely, there were significantly fewer TUNEL-positive apoptotic cells in the infarcted myocardium in the unloading group than in the loading group 3 1, B). This was possibly attributable to the heart failure and and 7 days after infarction P (.01, Figure 3, B). followed compensative hypertrophy of the surviving cardiomyocytes in the loading heart. Quantitative analysis Cardiac Stem Cells and SDF-1 Expression in the showed that the wall thickness of the infarcted left ventricle Infarcted Hearts was significantly greater but the area of infarction was The recruitment of cardiac stem cells was also detected by significantly smaller in the unloading group than in the immunostaining analysis 3 and 7 days after infarction. The loading group (P.05, Figure 1, D and F). c-kit- and Sca-1-positive stem cells were observed most 1054 The Journal of Thoracic and Cardiovascular Surgery April 2007

5 Suzuki et al Surgery for Acquired Cardiovascular Disease Figure 3. The apoptosis of cells in the injured heart. A, Representative photograph of the apoptotic cells. TUNEL-positive cells were labeled by green (left), and nuclei were stained by DAPI (blue, middle). Scale bars: 20 m. B, Quantitative counting of TUNEL-positive cells with a myocyte-specific morphologic structure revealed significantly more apoptotic cells in the loading group than in the unloading group 3 and 7 days after infarction. frequently in the border area of the infarcted myocardium (Figure 4, A and Figure 5, A). Our quantitative data showed that there were significantly more c-kit-positive stem cells in the border area of the infarcted myocardium in the unloading group than in the loading group on day 7 (P.05) but not on day P 3 (.28, Figure 4, B). Similarly, there were more Sca-1-positive stem cells in the unloading group than in the loading group on day 7 (P.05) but only a tendency was seen on day P 3 (.10, Figure 5, B). Immunostaining analysis revealed that the expression of SDF-1 was localized mainly in the border area of the infarcted myocardium and that the expression of SDF-1 was relatively stronger in the unloading heart than in the loading heart (Figure 6). Discussion Studies have demonstrated that recovery of cardiac function can be achieved by LVAD support, reducing the LV chamber size, increasing contractility of the cardiomyo - cytes, 19 20,21 normalizing the cardiac extracellular matrix, improving the expression of individual genes, and depressing cell apoptosis. 25 Although reverse remodeling and normalization of the neurohormonal milieu under unloading conditions with LVAD support is well documented, the role of hemodynamic loading on the activity of myocytes and cardiac stem cells is still unclear. In this study, we investigated how hemodynamic loading affects self-repair of the injured heart and examined the relative cellular and molecular mechanisms. To establish an LV hemodynamic unloading model in mice, we transplanted heterotopically an acute infarcted heart into the abdomen of another healthy mouse, with anastomosis of the donor ascending aorta to the recipient abdominal aorta and the donor pulmonary artery to the recipient inferior vena cava. 15 In the donor heart, blood from the abdominal aorta of the recipient mouse retroperfused into the coronary arteries of the donor heart, then drained into the right atrium, and entered the right ventricle of the donor heart, finally being ejected into the inferior vena cava of the recipient mouse. Thus, there was no hemodynamic loading in the infarcted left ventricle, but coronary perfusion was sustained. This condition is similar to that created by LVAD support. 26,27 We found a smaller area of infarction and greater wall thickness in the unloading heart than in the normal loading heart. Moreover, we observed distinctly that layers of cardiomyocytes remained in the endocardium of the unloading heart 28 days after infarction, but this was not observed in The Journal of Thoracic and Cardiovascular Surgery Volume 133, Number

6 Surgery for Acquired Cardiovascular Disease Suzuki et al Figure 4. The c-kit-positive stem cells in the injured heart. A, Representative photograph of the c-kit-positive stem cells in the infarcted heart. The c-kit-positive cells were stained by red (left), and nuclei were stained by DAPI (blue, middle). Scale bars: 20 m. B, Quantitative analysis revealed significantly more c-kit-positive cells in the loading group than in the unloading group 7 days after infarction. the loading infarcted heart. The ventricular weight was also increased significantly in the loading heart 28 days after infarction. Although we did not measure or compare the size of the cardiomyocytes, the increased ventricular weight might be related to the hypertrophy of the cardiomyocytes in response to heart failure. All of these findings suggest that the self-repair of the infarcted hearts was better under hemodynamic unloading conditions than under loading conditions. The fact that more proliferating cells but fewer apoptotic cells were observed in the unloading heart than in the loading heart 3 and 7 days postinfarction indicates that hemodynamic unloading increases the proliferation activity but decreases the apoptosis of cells in the infarcted heart. There were also more proliferating cells than apoptotic cells in the unloading heart, at a ratio of about 2 proliferating cells to 3 apoptotic cells per high-power field, but it was reversed in the loading heart, with a ratio of about 3 proliferating cells to 1.5 apoptotic cells per high-power field. According to our data, the balance between proliferation (regeneration) and apoptosis (loss) of cells was positive in the unloading heart but negative in the loading heart. Although many other factors need to be taken into consideration in the balance of regeneration and loss of myocytes, self-repair of the injured heart may occur under hemodynamic unloading conditions. This evidence may explain why obvious self-regeneration was not observed clinically in the injured heart under LV hemodynamic loading but cardiac function recovered frequently in the failing heart under LVAD support However, we counted about 300 nuclei per high-power field, so the proportion of Ki-67- positive cells would be about 1.0%. The proportion of Ki67-positive cells was comparable with the acute infarcted human heart. 3 As the level of proliferating cells is relative low, the increased proliferation and the decreased apoptosis of cells under LVAD support should contribute in a limited manner to repair the heart after infarction. We also found significantly more c-kit- and Sca-1-positive stem cells in the unloading heart than in the loading heart, although we could not identify the origination and fate of these stem cells, so we were unable to ascertain if they were heart-specific endogenous precursors or bone marrow derived stem cells. We do not know if these stem cells will differentiate and mature into myocytes for functional myocardial repair, although we previously found evidence that c-kit- and Sca-1-positive stem cells in the heart originate from bone marrow (data not shown). Thus, it is possible that the increased expression of SDF-1 in the unloading heart will induce the recruitment of stem cells 1056 The Journal of Thoracic and Cardiovascular Surgery April 2007

7 Suzuki et al Surgery for Acquired Cardiovascular Disease Figure 5. Sca-1-positive stem cells in the injured heart. A, Representative photograph of the Sca-1-positive cells in the infarcted heart. The Sca-1-positive cells were stained by red (left), and nuclei were stained by DAPI (blue, middle). Scales bars: 20 m. B, Quantitative analysis revealed significantly more Sca-1-positive cells in the loading group than in the unloading group 7 days after infarction. from bone marrow into the injured heart for myocardial repair. 28 Although we focused only on the mechanisms of myocardial repair in the turnover of myocytes and cardiac stem cells in the present study, previous investigations have found complex changes in the milium of the heart after LVAD support. These changes include a decrease in wall tension (pressure stress), improvement of coronary flow, Figure 6. The expression of SDF-1 in the heart 3 and 7 days after infarction. Immunostaining analysis revealed that the expression of SDF-1 was relatively weak in the loading heart (A); however, intensive expression of SDF-1 was observed in the unloading heart, especially in the border area of the infarcted myocardium. The Journal of Thoracic and Cardiovascular Surgery Volume 133, Number

8 Surgery for Acquired Cardiovascular Disease Suzuki et al reduction in lymphocyte infiltration and inflammatory cytokines, and normalization of the extracellular matrix. Thus, it is possible that the friendly milium in the unloading heart favors the survival and proliferation of myocytes and improves the survival, proliferation, differentiation, and maturation of cardiac stem cells for myocardial repair. The limitation of this study lies in the fact that the unloading model we used is not the same as that used for LVAD implantation. Moreover, we did not compare the recovery of LV function in the loading and unloading hearts; therefore, our data need to be confirmed in a largeanimal model and in clinical trials. Nevertheless, the results of this study provide the first evidence that hemodynamic unloading creates a positive balance between the regeneration and loss of myocytes in the injured heart by increasing cell proliferation, inhibiting cell apoptosis, and improving stem-cell recruitment. Accordingly, reducing hemodynamic loading may be a new strategy to assist self-regeneration of the injured heart. References 1. Zak R. Development and proliferative capacity of cardiac muscle cells. Circ Res. 1974;35(suppl II): Kajstura J, Leri A, Finato N, Di Loreto C, Beltrami CA, Anversa P. Myocyte proliferation in end-stage cardiac failure in humans. Proc Natl Acad Sci USA.1998;95: Beltrami AP, Urbanek K, Kajstura J, et al. Evidence that human cardiac myocytes divide after myocardial infarction. N Engl J Med. 2001;344: Oh H, Bradfute SB, Gallardo TD, et al. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci USA. 2003;100: Beltrami AP, Barlucchi L, Torella D, et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell. 2003;114: Laugwitz KL, Moretti A, Lam J, et al. Postnatal isl1 cardioblasts enter fully differentiated cardiomyocyte lineages. Nature. 2005;433: Matsuura K, Nagai T, Nishigaki N, et al. Adult cardiac Sca-1-positive cells differentiate into beating cardiomyocytes. J Biol Chem. 2004; 279: Vanderheyden M, Paulus WJ, Voss M, et al. Myocardial cytokine gene expression is higher in aortic stenosis than in idiopathic dilated cardiomyopathy. Heart. 2005;91: Olivetti G, Abbi R, Quaini F, et al. Apoptosis in the failing human heart. N Engl J Med. 1997;336: Muller J, Wallukat G, Weng YG, et al. Weaning from mechanical cardiac support in patients with idiopathic dilated cardiomyopathy. Circulation. 1997;96: Frazier OH, Myers TJ. Left ventricular assist system as a bridge to myocardial recovery. Ann Thorac Surg. 1999;68: Simon MA, Kormos RL, Murali S, et al. Myocardial recovery using ventricular assist devices: prevalence, clinical characteristics, and outcomes. Circulation. 2005;112(9 suppl):i Dang NC, Topkara VK, Leacche M, John R, Byrne JG, Naka Y. Left ventricular assist device implantation after acute anterior wall myocardial infarction and cardiogenic shock: a two-center study. J Thorac Cardiovasc Surg. 2005;130: Li TS, Hayashi M, Ito H, et al. Regeneration of infarcted myocardium by intramyocardial implantation of ex vivo transforming growth factor-beta-preprogrammed bone marrow stem cells. Circulation. 2005;111: Hamano K, Rawsthorne MA, Bushell AR, Morris PJ, Wood KJ. Evidence that the continued presence of the organ graft and not peripheral donor microchimerism is essential for maintenance of tolerance to alloantigen in vivo in anti-cd4 treated recipients. Transplantation. 1996;62: Levin HR, Oz MC, Chen JM, Packer M, Rose EA, Burkhoff D. Reversal of chronic ventricular dilation in patients with end-stage cardiomyopathy by prolonged mechanical unloading. Circulation. 1995;91: McCarthy PM, Nakatani S, Vargo R, et al. Structural and left ventricular histologic changes after implantable LVAD insertion. Ann Thorac Surg. 1995;59: Barbone A, Holmes JW, Heerdt PM, et al. Comparison of right and left ventricular responses to left ventricular assist device support in patients with severe heart failure: a primary role of mechanical unloading underlying reverse remodeling. Circulation. 2001;104: Dipla K, Mattiello JA, Jeevanandam V, Houser SR, Margulies KB. Myocyte recovery after mechanical circulatory support in humans with end-stage heart failure. Circulation. 1998;97: McGowan BS, Scott CB, Mu A, McCormick RJ, Thomas DP, Margulies KB. Unloading-induced remodeling in the normal and hypertrophic left ventricle. Am J Physiol Heart Circ Physiol. 2003;284: H Klotz S, Foronjy RF, Dickstein ML, et al. Mechanical unloading during left ventricular assist device support increases left ventricular collagen cross-linking and myocardial stiffness. Circulation. 2005; 112: Blaxall BC, Tschannen-Moran BM, Milano CA, Koch WJ. Differential gene expression and genomic patient stratification following left ventricular assist device support. J Am Coll Cardiol. 2003;41: Heerdt PM, Holmes JW, Cai B, et al. Chronic unloading by left ventricular assist device reverses contractile dysfunction and alters gene expression in end-stage heart failure. Circulation. 2000;102: Bartling B, Milting H, Schumann H, et al. Myocardial gene expression of regulators of myocyte apoptosis and myocyte calcium homeostasis during hemodynamic unloading by ventricular assist devices in patients with end-stage heart failure. Circulation. 1999;100(19 suppl): II Hattori T. Effect of mechanical assist devices for ischemic myocardial damage-cardiomyocyte apoptosis and TNF-alpha. Ann Thorac Cardiovasc Surg. 2003;9: Ito K, Nakayama M, Hasan F, Yan X, Schneider MD, Lorell BH. Contractile reserve and calcium regulation are depressed in myocytes from chronically unloaded hearts. Circulation. 2003;107: Tevaearai HT, Walton GB, Eckhart AD, Keys JR, Koch WJ. Heterotopic transplantation as a model to study functional recovery of unloaded failing hearts. J Thorac Cardiovasc Surg. 2002;124: Wojakowski W, Tendera M, Michalowska A, et al. Mobilization of CD34/CXCR4, CD34/CD117, c-met stem cells, and mononuclear cells expressing early cardiac, muscle, and endothelial markers into peripheral blood in patients with acute myocardial infarction. Circulation. 2004;110: Wohlschlaeger J, Schmitz KJ, Schmid C, et al. Reverse remodeling following insertion of left ventricular assist devices (LVAD): a review of the morphological and molecular changes. Cardiovasc Res. 2005; 68: Sukehiro S, Flameng W. Effects of left ventricular assist for cardiogenic shock on cardiac function and organ blood flow distribution. Ann Thorac Surg. 1990;50: Ankersmit HJ, Edwards NM, Schuster M, et al. Quantitative changes in T-cell populations after left ventricular assist device implantation: relationship to T-cell apoptosis and soluble CD95. Circulation. 1999; 100(19 suppl):ii The Journal of Thoracic and Cardiovascular Surgery April 2007