Download Online Appendix for the following October 20 JACC article

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