ONLINE APPENDIX
Roles of Angiotensin Peptides and Recombinant Human ACE2 in Heart Failure
Ratnadeep Basu, MD, PhD1, 2, Marko Poglitsch, PhD3, Haran Yogasundaram, MD1,2,
Jissy Thomas, MN2, Brian H. Rowe, MD4 and Gavin Y. Oudit, MD, PhD1, 2
1Division of Cardiology, Department of Medicine, 2Mazankowski Alberta Heart Institute, University of
Alberta, Edmonton, Canada, 3Attoquant Diagnostics, Vienna, Austria, and 4Department of Emergency
Medicine and School of Public Health, University of Alberta,
Edmonton, Canada
METHODS
Prior to phlebotomy, all study participants rested in a supine position for approximately 45 min
to elude any variation due to different body positions. Blood was collected in pre-labeled tubes loaded
with an angiotensin-stabilizing protease inhibitor cocktail, lithium heparin, or ethylenediaminetetraacetic
acid (EDTA). Tubes containing blood were shaken thoroughly for homogenous mixture of respective
contents prior centrifugation at 3000 x g for 10 min at 4 C. Supernatant plasma was collected in prelabeled aliquots and flash frozen in liquid nitrogen before being stored at -80 C for further analyses.
Circulating Peptide Measurement and Analysis. Circulating Ang peptide concentrations were
determined by mass spectrometry (MS) using plasma samples collected in the presence of an inhibitor
cocktail completely blocking angiotensin metabolism, containing broad spectrum inhibitors against
metalloproteases (EDTA, 1,10-phenanthroline), aspartic proteases (pepstatin A), cysteine proteases (phydroxymercuribenzoic acid), serine proteases (AEBSF), and specific inhibitors for renin and
aminopeptidases A and N to a final concentration of 5% v/v (Attoquant Diagnostics, Vienna, Austria).
Supplemental Table 1. Sample preparation recovery rates and lower limits of quantification of
the individual angiotensin peptides.
Recovery rates shown in percentage for individual peptide; LLOQ=Lower limits of detection measured
in pg/ml for circulating and equilibrium states.
Supplemental Table 2. Additional plasma angiotensin peptides profiled using liquid chromatography-mass
spectrometry.
N=number of patients per group; HC=healthy control; CHF-ACEI=Chronic Heart Failure treated with ACE
inhibitor; AHF=Acute Heart Failure; median values of peptide levels in pg/ml reported with interquartile
range (IQR) shown in parenthesis; C=Circulating levels; EQ=Equilibrium levels; NND=Number of samples
below lower limits of quantification.
Supplemental Figure 1. Circulating levels and equilibrium levels of Ang 1-10 (A), Ang II (B), Ang 17 (C) and the Ang 1-7/Ang II ratio (D) shown in box and whisker plots in relation to ejection fraction in
the entire cohort of patients with heart failure.
Supplemental Figure 2. Recombinant human ACE2 treatment increases the Ang 1-7/Ang II ratio in
equilibrium analysis of plasma samples of CHF patients treated with an angiotensin receptor blocker
(ARB). Illustration of 10 Ang peptides (numbered in parenthesis along with concentrations in pg/ml;
diameter of spheres are proportional to the concentration of the representative Ang peptide) in plasma of
CHF patients treated with ARB and analyzed in equilibrium before (pre) and after (post) the addition of 5
μg/ml rhACE2 (n=17) (A). Equilibrium plasma levels of Ang 1-10 (B), Ang II (C), Ang 1-7 (D) and Ang 17/Ang II ratio (E). **p <0.01
Supplemental Figure 3. Western blot analysis (A) and quantification (B) showing comparable protein
levels of the Mas receptor in hearts with NFC (n=6) and DCM (n=6). R.R.=relative ratio.