Download on line supplement to: mineralocorticoid signalling in transition to

Shapiro et al; Hypertension MS # 099010 Supplemental methods
ON LINE SUPPLEMENT TO: MINERALOCORTICOID SIGNALLING IN
TRANSITION TO HEART FAILURE WITH NORMAL EJECTION FRACTION
Brian P. Shapiro, MD1
Theophilus E. Owan, MD2
Selma Mohammed, MBBS1
Martina Kruger, PhD3
Wolfgang A. Linke, PhD3
John C Burnett, Jr, MD1
Margaret M. Redfield, MD1
*Drs. Shapiro and Owan contributed equally to this study.
1
Division of Cardiovascular Diseases, Mayo Clinic and Foundation, Rochester, MN, Unites
States, 55905; 2Division of Cardiology, University of Utah; 3Physiology and Biophysics Unit,
University of Muenster, Schlossplatz 5, D-48149 Muenster, Germany
Short Title: Aldosterone and HFnlEF
Address for correspondence:
Margaret M. Redfield, MD
Mayo Cardiorenal Research Laboratory
200 First Street SW
Rochester, MN 55905
Tel. (507) 284-1281
Fax (507) 266-4710 E-mail: [email protected]
Shapiro et al; Hypertension MS # 099010 Supplemental methods
Supplemental methods:
Acute Hemodynamic Study
Dogs were ventilated with supplemental oxygen, and given intravenous (IV) saline (3
ml/min) to replace insensible losses. Micromanometer catheters (Millar Instruments, Houston,
TX) were placed in the LV and ascending aorta. A pneumatic inferior vena cava (IVC) occluder,
LV short (SA) and long (LA) axis and aortic piezoelectric crystals (Sonometrics Corp, Ontario,
Canada), and an ascending aortic volumetric flow probe (Transonics, London, England) were
placed. Dogs were atrial paced at 10-20 bpm above sinus rate and received adrenergic blockage
with propranolol (2 mg/kg IV) and atropine (1.0 mg IV).
Following sacrifice, LV mass was recorded and sections of the LV and aorta were placed
in formalin or flash-frozen and stored at -80C.
Data were acquired at 250 Hz and analyzed with commercially available software
(Sonometrics, Ontario, Canada) as previously described1
LV systolic (ESV) and diastolic (EDV) volumes (π/6*SA2*LA) were used to calculate
SV and CO. Acute IVC occlusion was used to define the end-systolic pressure (ESP) volume
(ESV) relationship (ESPVR; ESP=Ees(ESV-V0)) and the end-diastolic pressure (EDP) volume
relationship (EDPVR; EDP=e(*EDV)) where  represents the diastolic stiffness coefficient and
, the curve fitting constant. To account for nonlinear covariance of  and , EDV at a common
EDP of 20 mmHg was calculated (EDV20 = [ln (20/α)]/β) where a lower EDV20 reflects
increased diastolic stiffness2-4.
Effective arterial elastance (Ea), was calculated as ESP/SV. Systemic vascular resistance
(SVR) was calculated by mean arterial pressure (MAP)/CO and expressed as dyne sec/cm5. The
characteristic aortic impedance (Zo) was determined by Fourier analysis of aortic pressure and
Shapiro et al; Hypertension MS # 099010 Supplemental methods
flow from the mean of impedance moduli from 2-5 Hz and expressed as dyne sec/cm5 5.
Systemic arterial compliance (SAC) was calculated by the method of Liu et al where SAC is
assessed during diastole as ∫AoP (from PDN to Pd) /SVR (PDN-Pd) where PDN is aortic P at the
dicrotic notch and Pd is aortic pressure at end-diastole6.
Neurohumoral Analysis
Blood samples were collected prior to renal wrap and at week eight. Procollagen III was
assessed by ELISA (Orion Diagnostica, Finland). Plasma angiotensin II (AII), aldosterone, and
plasma and LV brain natriuretic peptide (BNP) were measured by radioimmunoassay (RIA) as
previously described7. LV BNP levels were normalized to protein content determined by the
Lowry method8. Creatinine concentration was determined by absorbance spectroscopy.
Transforming growth factor-1 (TGF-1) content was measured according to a commercially
available ELISA kit (R&D Systems, Minneapolis, MN) and by Western blot analysis as
previously described9. Signals were analyzed on ImageJ software (National Institutes of Health,
Bethesda, Maryland) and expressed as an integrated density from the ratio of the actin signal.
Total Collagen Content and Cross-Linking
Total collagen and collagen solubility (pepsin digestion with 1 mg/ml of pepsin x 24
hours at 37C) from the LV and aorta were measured by the hydroxyproline assay10.
References
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Shapiro et al; Hypertension MS # 099010 Supplemental methods
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