<i>PDE5 is not detected in cardiac LV and RV lysates from canines</i>.

<p>PDE5 was not detected in tissue lysates from the LV or RV in young normal (Normal) or old hypertensive canines (Old HTN) with diastolic dysfunction using the rabbit polyclonal anti-PDE5 antibody provided by Joseph A Beavo, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118664#pone.0118664.ref026" target="_blank">26</a>]. Bovine lung is used as a positive control.</p

Human Samples/Patient Characteristics.

<p>Human Samples/Patient Characteristics.</p

<i>PKG is detected in both canine and human LV samples</i>.

<p>(A) PKG is detected in LV tissue lysates in young normal (Normal), old hypertensive canines (Old HTN) with diastolic dysfunction, as well as bovine lung. (B) PKG is detected by immunoblotting of human LV samples (heart failure (n = 6) and controls (n = 8), as well as bovine lung. Upper blot—HF 1-3, Control 1-4 and lower blot—HF 4-6, Control 5-8 (refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118664#pone.0118664.t001" target="_blank">Table 1</a>).</p

<i>PDE5 is not detected in LV samples from human controls or patients with heart failure</i>.

<p>(A) PDE5 is not detected by immunoblotting of human LV samples (heart failure (n = 6) and controls (n = 8); bovine lung is used as a positive control (rabbit polyclonal anti-PDE5 antibody provided by Joseph A Beavo, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118664#pone.0118664.ref026" target="_blank">26</a>]). Upper blot—HF 1–3, Control 1–4 and lower blot—HF 4–6, Control 5-8 (refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118664#pone.0118664.t001" target="_blank">Table 1</a>). (B) Western blots of 2-D gels with the anti-PDE5 antibody (Cell Signaling) demonstrate that PDE5 is detected in the bovine lung as 2 isoelectric variants, while PDE5 is not detected in cardiac tissue lysates of human LV samples (control (Control 2)) and heart failure (HF1)).</p

PDE5 is not detected in murine LV.

<p>(A) Western blots of LV samples from the murine demonstrate that PDE5 is detected in samples from the lung used as a positive control. For LV samples from normal (control), heart failure and compensated hypertrophy, there is non-specific binding of the anti-PDE5 antibody from Santa Cruz at a MW near the positive control, while PDE5 is not detected with the anti-PDE5 antibody from Cell Signaling or the rabbit polyclonal anti-PDE5 antibody provided by Joseph A Beavo, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118664#pone.0118664.ref026" target="_blank">26</a>]. (B) Western blots of 2-D gels with the anti-PDE5 antibody (Cell Signaling) demonstrate that PDE5 is detected in the lung as 2 isoelectric variants, while PDE5 is not detected in cardiac tissue lysates (normal mice). However in cardiac tissue lysates, the anti-PDE5 antibody from Santa Cruz demonstrates non-specific binding.</p

Effect of rapamycin on cardiomyocyte cross-sectional area in HF.

<p>Representative left ventricular myocardial sections and grouped data for cardiomyocyte area in SHAM operated (1±0.04, n = 5), placebo treated HF (1.40±0.05, n = 5) and 8 mg/kg/day PO rapamycin treated HF mice (1.21±0.05, n = 6). Data are mean ± SEM. †: <i>P</i><0.05 vs SHAM, ‡: <i>P</i><0.05 vs. placebo treated HF mice.</p

Effect of rapamycin on myocardial mTOR signaling in normal mice.

<p>Western blots and grouped data are shown for S6K1 and Thr389 phosphorylated S6K1 (A), or S6 and Ser235/236 phosphorylated S6 (B) in rapamycin (8 mg/kg/day PO, n = 4) versus placebo treated normal mice (n = 4). Rapamycin treatment showed a strong trend towards decreasing total S6K1 expression, whereas Thr389 phosphorylated S6K1 and total and Ser235/236 phosphorylated S6 were all significantly decreased. Data are mean ± SEM. †: <i>P</i><0.05 vs. placebo.</p

Effect of rapamycin and/or losartan in TAC mice with heart failure (TAC-HF).

<p>BW: body weight; EF: ejection fraction; HW: heart weight; LV: left ventricular; LVEDD: Left ventricular end diastolic dimension; LVSP: LV systolic pressure. †: <i>P</i><0.05 vs TAC-HF + placebo and ‡: <i>P</i><0.05 vs TAC-HF + Rapamycin.</p

Serum rapamycin levels in response to dosing and administration routes.

<p>Mice were dosed with 2, 4, 5 or 8/kg/day rapamycin through intraperitoneal (IP) or oral (PO) administration routes. Serum levels from 2 (n = 5), 5 (n = 6) and 8 (n = 8) mg/kg/day PO administration were dose dependent, with the latter approaching serum levels achieved with 2 mg/kg/day IP administration (n = 5). Serum rapamycin levels for HF mice given 8 mg/kg/day PO (n = 13) dosing were not different than that for normal mice, whereas 4 mg/kg/day PO (n = 6) showed reduced serum levels as expected. Data are mean ± SEM. †: <i>P</i><0.05 vs. 2 mg/kg/day IP.</p

Effect of rapamycin on autophagy in HF.

<p>Representative Western blots and grouped data for LC3B-II expression (bottom band) in myocardium from SHAM operated (n = 6), placebo treated HF (n = 6) and 8 mg/kg/day PO rapamycin treated HF mice (n = 6). Std: A standard LV homogenate sample for reliable comparison across gels. Data are mean ± SEM. †: <i>P</i><0.05 vs SHAM, ‡: <i>P</i><0.05 vs. placebo treated HF mice.</p