Heart Rate and Diastolic Parameters.

<p>Data are expressed as mean ± SEM. Emax  =  maximum early transmitral filling velocity; E dec time  =  early-filling deceleration time; E dec slope  =  early-filling deceleration slope; e' =  early mitral annular velocity; E/e'  =  early transmitral filling velocity-to-mitral annular velocity ratio.</p

Uptake of Ang-(1–12) by neonatal cardiac myocytes.

<p>Time dependent increase in cellular uptake of ¹²⁵I-Ang-(1–12) in cultured neonatal SHR cardiac myocytes (solid line) as compared to WKY (dotted line) in the presence of all RAS enzymes inhibitors. Cellular internalization of ¹²⁵I-Ang-(1–12) is expressed as fmol⁻¹ x mg⁻¹ protein. Each value represents the mean ± SE of three or more independent assays. In these experiments, WKY and SHR myocytes were treated with all inhibitor cocktail containing lisinopril (ACE inhibitor), SCH39370 (NEP inhibitor), MLN-4760 (ACE2 inhibitor), and chymostatin (chymase inhibitor), peptidase inhibitors (amastatin, bestatin & benzyl succinate), and PCMB each added at a dose of 10 µM. *Significantly different (<i>P</i><0.001) from the mean of the corresponding time point of WKY.</p

Effect of inhibitors on Ang-(1–12) metabolism in cardiac myocytes.

<p>Characterization by high pressure liquid chromatography of cellular ¹²⁵I-Ang-(1–12) products internalized by 24-h serum deprived cultured neonatal WKY (A and B) and SHR (C and D) cardiac myocytes in the presence of all inhibitors cocktail (containing lisinopril, SCH39370, MLN-4760, chymostatin, amastatin, bestatin, benzyl succinate and PCMB) and in the absence of RAS inhibitors (containing only amastatin, bestatin, benzyl succinate & PCMB) each added at a dose of 10 µM. Before adding the ¹²⁵I-Ang-(1–12), the cultured cardiac myocytes were pre-incubated with inhibitors for 15 min at 37°C. The arrow indicates the peak area of ¹²⁵I-Ang-(1–12).</p

Outline of enzyme inhibitors employed in the experiments.

<p>Abbreviations as in text.</p

Sequence of PCR Primers.

<p>S16; GPER, G protein-coupled estrogen receptor; BNP, brain natriuretic peptide.</p

Cardiac Geometry and Systolic Parameters.

<p>Data are expressed as mean ± SEM. LV mass  =  left ventricular mass; LVEDd  =  left ventricular end-diastolic dimension; LVESd  =  left ventricular end-systolic dimension; RWT  =  relative wall thickness; %FS  =  percent fractional shortening.</p

Enzyme activities in WKY and SHR cardiac myocytes.

<p>Relative contributions of each enzyme to the ¹²⁵I-Ang-(1–12) metabolism by WKY and SHR cardiac myocytes documents higher enzymatic activity in SHR. These enzymes activity were calculated based on peptide fractions generated from Ang-(1–12) under various RAS inhibitors condition used (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015759#s4" target="_blank">Material and Methods</a>). Abbreviations are: ACE, angiotensin converting enzyme; NEP, neprilysin, ACE2, angiotensin converting enzyme 2. Values are means ± SE. *, p<0.05.</p

G1 treatment for two weeks does not affect salt-associated increases in blood pressure.

<p>Tail-cuff systolic blood pressure in conscious female mRen2.Lewis rats fed a normal salt (NS) or high salt (HS) diet over the course of the experiment. Arrow indicates time of pump insertion for 14-day delivery of either G-1 or vehicle (VEH). Values are mean ± SEM; * P<0.05 compared to NS (Salt effect).</p

Comparative effects of selective enzyme inhibition on ¹²⁵I-Ang-(1–12) metabolism in medium of cultured neonatal wky and shr cardiac myocytes.

<p>Values are expressed as % (Mean ± SE) of Ang-(1–12) unmetabolized* and Ang peptides (%) generated from ¹²⁵I-Ang-(1–12) in the cultured medium incubated for 60 min at 37°C to WKY or SHR myocytes with or without the presence of RAS inhibitors. <i>No inhibitors group</i>: Only aminopeptidases inhibitors (amastatin & bestatin) and carboxypeptidases inhibitor (benzyl succinate); <i>All inhibitors group</i>: Aminopeptidases and carboxypeptidases inhibitors + RAS inhibitors (lisinopril, SCH39370, MLN-4760 & chymostatin); <i>Minus RAS inhibitor groups</i>: One of the RAS inhibitor (lisinopril/SCH39370/MLN-4760/chymostatin) omitted at a time from <i>all inhibitors group</i>. All values are expressed as (%) and are the average of three or more independent assays.</p><p>*Percent of ¹²⁵IAng-(1–12) parent control remained un-metabolized in the medium exposed to WKY or SHR myocytes for 60 min at 37°C.</p>a<p>Significantly different (P<0.05) in <i>No RAS inhibitors</i> group vs. corresponding WKY or SHR group of +<i>All RAS inhibitors</i>.</p>b<p>Significantly different (P<0.05) vs. corresponding group of +<i>All RAS inhibitors</i>.</p>c<p>Significantly different (P<0.05) vs. corresponding WKY.</p

Physical Characteristics of the Four Experimental Groups.

<p>Data are expressed as mean ± SEM. Body weight gain represents the percent change in weight from week 13 (prior to pump insertion) to week 15.</p