ONLINE SUPPLEMENTARY MATERIAL在线辅助材料.doc

Online Appendix for the following JACC article TITLE: A Micro-Ribonucleic Acid Signature Associated With Recovery From Assist Device Support in Two Groups of Patients With Severe Heart Failure AUTHORS: Ravi Ramani, MD, Deborah Vela, MD, Ana Segura, MD, Dennis McNamara, MD, Bonnie Lemster, MPH, Vishnupriya Samarendra, BS, Robert Kormos, MD, Yoshiya Toyoda, MD, Christian Bermudez, MD, O. H. Frazier, MD, Christine S. Moravec, PHD, John Gorcsan III, MD, Heinrich Taegtmeyer, MD, DPHIL, Charles F. McTiernan, PHD APPENDIX Methods Cardiac functional characterization. For the functional characterization of the Test cohort, we used echocardiographic automated border detection from mid-ventricular short-axis images and noninvasive arterial pressure to measure beat-to-beat responses in 2- to 5-minute trials of decreased device flow. An increase in stroke area, 40% increase in fractional area change, or a preload adjusted maximal power 4.0 mW/cm4 with low device flow were associated with successful device removal. Additional evaluation of recovery involved exercise physiology with gas exchange analysis and invasive hemodynamics by right heart catheterization, both with the VAD rate lowered. Right heart catheterization was performed using the same VAD flow reduction protocol as for echocardiography (1,2). The clinical assessment of heart failure etiology for each cohort was equally distributed in each group, as presented in Supplementary Table 1. Tissue collection and processing. LV free wall samples were collected in the operating room, immediately immersed in cold cardioplegia, flash frozen within 15 minutes, and stored at 80oC. A subset of Test cohort samples had tissue fixed (2% paraformaldehyde) and frozen for future microscopic analyses (3). All Validation cohort samples had tissue fixed in formalin (4). Screening real-time PCR array. PCR array was performed on VAD core samples obtained from 6 patients in each group of the Test cohort. RNA was isolated (Qiagen miREasy), enrichment for small RNAs (SA Biosciences RT2 qPCR-Grade miRNA Isolation Kit), copied to cDNA, and analyzed with a microRNA microarray platform (SA Biosciences MAH-3100, Genome-Wide Array) on an Applied Biosystems 7900HT instrument on sequential days. Array data analysis. Fluorescence data acquired with Applied Biosystems Sequence Detection Systems (SDS) version 2.2.2 software was analyzed through the SA Biosciences PCR Array Data Analysis Portal (/pcr/arrayanalysis.php). Two housekeeping assays, SNORD44 and U6, were used for normalization. Real-time PCR confirmation. Ten miRs identified as being differentially expressed between the Recovered (R) and Dependent (D) groups by the array analysis were examined by confirmatory PCR. A pool of miR-specific primers (Applied Biosystems Megaplex Pool A RT primers) and total RNA was used to generate cDNA via a reverse transcription (RT) reaction. The cDNA was then pre-amplifed (Applied Biosystems, Megaplex Pool A pre-amplification primers). The amplified cDNA was used in duplicate individual TaqMan PCR reactions (using the miR-specific primer sequences from the Megaplex pool set A, and RNU48 primers as internal control) on an Applied Biosystems ABI Prism 7000. The Ct method was used to calculate the relative expression (5), using RNU48 Ct as the reference transcript. Additional miRs (miRs 1, 21, 133a, 133b, and 208) of potential (15-24) were also analyzed in the pre-amplified pool cDNA. To determine the effect of mechanical unloading, miRs found to be differentially expressed between R and D groups, as well as the miRs listed above, were measured in the paired samples recovered from hearts at the time of VAD implantation and cardiac transplantation. Microscopic analysis. Test cohort samples from clinically matched R (n = 3) and D (n = 3) patients were cryosectioned and stained with FITC-labeled wheat germ agglutinin so as to outline cardiomyocytes, and DAPI to identify nuclei. Images were obtained by fluorescence microscopy (20). The mean cross-sectional area of cardiomyocytes with identifiable nuclei was calculated from measurements of 25 randomly selected myocytes in 5 sections from each sample (3). From the Validation cohort, R (n = 7) and D (n = 7) samples (paraffin embedded) were serially sectioned, stained with hematoxylin and eosin, images obtained at 20 magnification and myocyte diameters obtained and analyzed as previously described (4). Measurements were obtained on at least 10 randomly selected myocytes in 5 sections per sample. References 1. Simon MA, Kormos RL, Murali S, et al. Myocardial recovery using ventricular assist devices: prevalence, clinical characteristics, and outcomes. Circulation 2005;112:I32-6. 2. Gorcsan J III, Severyn D, Murali S, Kormos RL. Non-invasive assessment of myocardial recovery on chronic left ventricular assist device: results associated with successful device removal. J Heart Lung Transplant 2003;22:1304-13. 3. McGaffin KR, Sun CK, Rager JJ, et al. Leptin signalling reduces the severity of cardiac dysfunction and remodelling after chronic ischaemic injury. Cardiovasc Res 2008;77:54-63. 4. Segura AM, Frazier OH, Demirozu Z, Buja LM. Histopathologic correlates of myocardial improvement in patients supported by a left ventricular assist device. Cardiova