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Online Appendix for the following JACC article TITLE: Impact of Mechanical Unloading on Microvasculature and Associated Central Remodeling Features of the Failing Human Heart AUTHORS: Stavros G. Drakos, MD, Abdallah G. Kfoury, MD, Elizabeth H. Hammond, MD, Bruce B. Reid, MD, Monica P. Revelo, MD, PhD, Brad Y. Rasmusson, MD, Kevin J. Whitehead, MD, Mohamed E. Salama, MD, Craig H. Selzman, MD, Josef Stehlik, MD, Michael R. Bristow, MD, PhD, Dale G. Renlund, MD, Dean Y. Li, MD, PhD APPENDIX A. Supplemental Methods Myocardial tissue immediately after its excision in the operating room was fixed in 10% buffered formalin for 24 hours and dehydrated in increasing concentrations of alcohol, then cleared through xylene and subsequently embedded in paraffin. The tissue sections were cut in 4-μm sections, collected and mounted on glass slides and prepared for various histochemical stains and immunohistochemistry. 1. Masson’s Trichrome Masson’s trichrome stain was used for collagen content evaluation. The stain was performed as previously described (1). 2. Periodic acid Schiff stain reaction (PAS) – cardiac myocyte size evaluation The reaction is based on oxidation of certain tissue elements to aldehydes by periodic acid. The stain was performed as previously described (2). PAS was selected for cardiomyocyte size evaluation because it offers sufficient cardiomyocyte basement membrane visualization (2). Cardiomyocytes (40x magnification) were accepted for size measurement if they met the following criteria: (a) cellular crosssections present (b) visible and round shaped nuclei located close to the cell center and (c) intact cellular basement membranes. The cross section area (CSA) of 100 selected cardiomyocytes per patient sample was calculated by our digital histopathology system and then averaged. 3. Combination of PAS with diastase digestion (PASD) and standard PAS The combination of PAS and PAS with diastase (PASD) was used for demonstration of glycogen content. PAS stains magenta the cardiomyocyte glycogen stores but it also stains with the same color additional tissue elements (neutral mucosubstances, epithelial sulfomucins and sialomucins) (2). PASD reaction digests glycogen with a pretreatment of the tissue with α-amylase (depolymerizes glycogen) and as a result of this any magenta staining is solely attributed to the other tissue elements described above but not to glycogen (2). Consequently, glycogen stores can be evaluated by digital subtraction of the images derived from two paired adjacent slides images (stained with PAS and PASD each) as described in the digital microscopy section of methods. PASD reaction was performed with a 20-min pretreatment of the tissue sections with diastase solution 0.5% (enzyme α-amylase 0.25g, distilled water 50ml). 4. Immunohistochemistry Microvasculature evaluation was performed with immunostaining for endothelial cell protein CD34 and endothelial activation marker MHC-2. Immunohistochemistry experiments were performed using a peroxidase- conjugated streptavidin-biotin system and diaminobenzidine as a substrate. For preliminary experiments, myocardial samples were stained at varying concentrations of anti-CD34 (mouse monoclonal antibody, Dako, Carpinteria, CA) and anti-MHC2 (mouse monoclonal antibody, Abcam Inc, Cambridge, MA). Peak staining occurred at 1/ 50 for CD34 immunostaining and at 1 / 100 for MHC-2. These concentrations of primary antibodies were used for all subsequent studies. To achieve high degree of reproducibility we avoided manual staining. Histochemical stains were performed using the automatic staining system Artisan Special Stains System (Dako, Carpinteria, CA) and the immunohistochemistry experiments were performed on the Autostainer (Dako, Carpinteria, CA). 5. Ultrastructural evaluation Tissue for electron microscopy was fixed in 4ºC Karnovsky’s fixative immediately after its excision in the operating room and subsequently was embedded in Epon 812. One micron-thick sections were stained with toluidine blue and representative sections (both longitudinal and cross section of myocardium) were examined. Analysis was performed on a JEM 1010 electron microscope (Jeol, Peabody, Mass). Tissue was examined according to an ultrastructural classification scheme which included the following parameters (3-6): - cardiomyocyte hypertrophy (myocyte size, nuclear enlargement/irregular shapes, increased numbers of mitochondria, widened and convoluted intercalated disks), - cardiomyocyte degeneration (myofilament loss, abnormalities of mitochondrial size and structure, aggregation of glycogen in areas of myofilament loss, aggregation of abnormal Z band material, accumulation of myelin figures and lipofuscin containing lysosomes, and dilatation of sarcotubular elements), - microvascular abnormalities (endothelial cell swelling, endothelial nuclear enlargement, laminated basal lamina accumulation, and increases in lumenal cytoplasmic processes and pinocytotic vesicle formation). These parameters were graded 0 (none) to 3+ (extensive). B. Supplemental Results Pertinent medications that the study population was treated with during the LVAD support period are shown in supplemental table 1. Supplemental table 1. Use of medications during left ventricular assist device support Medication N (%) ACE Inhibitor/ ARB (%) 2 (13) Beta blocker (%) 0 Aldosterone antagonist (%) 0 Calcium channel blocker (%) 6 (40) Diuretic (%) 8 (53) Digoxin (%) 4 (27) Statin (%) 0 Aspirin (%) 15 (100) Dipyridamole (%) 15 (100) Warfarin (%) 5 (33) ACE: angiotensin converting enzyme; ARB: angiotensin II receptor blocker; Correlations between variables We analyzed whether changes in pulmonary capillary wedge pressure were correlated with changes in structural parameters. Correlations among the various variables were evaluated using Pearson’s correlation coefficient. The increase in interstitial fibrosis correlated with the degree of pressure unloading as measured by reduction in pulmonary capillary wedge pressure (R = 0.7, p=0.009). No other significant correlations were identified. Endothelial cell activation To add further support to our structural and ultrastructural microvasculature findings immunohistochemical stains for the endothelial activation marker MHC-2 were performed. Compared to the pre LVAD patient samples the post LVAD expression of this marker was consistently found to be significantly increased in all of our patients (Supplemental Figure 1). Figure legend, supplemental figure 1. Immunostaining for endothelial activation marker MHC-2 (brown color). Representative figures of two paired patient samples (100x magnification), LVAD: left ventricular assist device, MHC-2: major histocompatibility complex class 2 Supplemental References 1. Carson FL. Connective and muscle tissue. In: Carson FL, ed. Histotechnology: a self-instructional text, Singapore: American Society for Clinical Pathology Press, 2007: 131-156 2. Carson FL. Carbohydrates and amyloid. In: Carson FL, ed. Histotechnology: a self-instructional text, Singapore: American Society for Clinical Pathology Press, 2007: 111-131 3. Hammond EH, Menlove RL, Anderson JL. Predictive value of immunofluorescence and electron microscopic evaluation of endomyocardial biopsies in the diagnosis and prognosis of myocarditis and idiopathic dilated cardiomyopathy. Am Heart J 1987;114:1055-65. 4. Hammond EH, Yowell RL. Utility of ultrastructural studies of cardiac biopsy specimens. Ultrastruct Pathol 1994;18:201-2. 5. She RC, Hammond EH. Utility of Immunofluorescence and Electron Microscopy in Endomyocardial Biopsies from Patients with Unexplained Heart Failure. Cardiovasc Pathol 2009, Jun 5 [Epub ahead of print] 6. Albala A, Fenoglio FF. Diagnostic electron microscopy of endomyocardial biopsies. In: Papadimitriou JM, Henderson DW, Spagnolo DV, editors. Diagnostic ultrastructure of non-neoplastic diseases. Churchill Livingstone, 1992; 254-263