y values were plotted as a function of reaction time and fitted by linear regression. The fit and the data were accepted when r.0.9. ACE activity was calculated by the equation: ACE activity = 2D, where S is PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19645759 the rate of observed decrease in optical density, k is the change in optical density upon the complete cleavage of 1 nmol of FAPGG, and D is the dilution of the serum. One unit of ACE activity represents 1 nmol of FAPGG hydrolysis per minute at 37 uC. Echocardiographic measurements Transthoracic echocardiography was performed using an Accuson Sequoia echocardiograph. Two dimensional and Doppler imaging was performed in standard parasternal and apical views. The left ventricular ejection fraction was measured by M mode left ventricular dimensional method. Preserved EF cutoff was.50%. Values for dP/dt were determined in patients with severe mitral regurgitation. Two experienced purchase TMS cardiologists unaware of the biochemical data performed the echocardiographic measurements. Blood sample collection Blood samples were collected by using a standard aseptic technique. Native blood was incubated for 60 minutes at room temperature. Serum fractions were separated by centrifugation and kept in a freezer until the measurements. Measurement of serum ACE2 activity The sACE2 activity measurement was performed using a specific quenched fluorescent substrate as previously described with some modifications . The reaction mixture contained 20 ml serum, 80 mL buffer and 100 ml sACE2-specific fluorescent substrate acetyl-Ala-Pro-Lys-OH . sACE2 activity was measured by fluorometric assay of the enzymatic cleavage of K from the fluorogenic substrate Mca-APK. The reaction buffer contained a protease inhibitor cocktail 10 mM Bestatin-hidrochloride, 10 mM Z-prolyl-prolinal, 5 mM Amastatin-hidrochloride, 10 mM Captopril and 5 mM NaCI, 100 mM ZnCI2, 75 mM TRIS HCI, pH 6.5. All chemicals were from Sigma if not stated otherwise. The reaction was performed in black 96-well microtiter plates. The assay was monitored continuously by measuring the increase in fluorescence Measurement of serum ACE concentration ACE concentration was 
determined using a Human ACE enzyme-linked immunosorbent assay Development System according to the manufacturer’s instruction, with minor modifications. In brief, enzymelinked immunosorbent assay plates were coated with capture antibody diluted to a working concentration of 80 ng/well in Dulbecco’s modified phosphatebuffered saline solution overnight at room temperature. The remaining binding sites were blocked with bovine serum albumin, 10 mg/mL, dissolved in DPBS. Human serum samples were diluted 100-fold in the same buffer and incubated with the immobilized primary antibodies for 2 hours. Capture antibody-bound ACE was labeled using a biotinylated detection antibody, 20 ng/well for 2 hours. Streptavidin-conjugated horseradish-peroxidase was added to the wells and incubated for 20 minutes. The immunocomplexes were detected Circulating ACE2 in Human Heart Failure with a chromogenic substrate solution containing 0.3 mg/mL TMB, 0.1 mM H2O2 and 50 mM acetic acid. Reaction was terminated by addition of 0.5 M HCl and was evaluated by measuring absorbance at 450 nm. ACE concentration was calculated using a calibration curve. The ACE concentration in the samples were measured at least three times to achieve a standard deviation of at most 15%. Serum ACE concentration was given as ng/mL of serum. Effects of biventricular pacing on echocardiographic p