Left ventricular function is reduced in rats following LAD ligation.

<p>Bar graph for LVEF (mean±SEM) in uninfarcted, control rats (n = 9), as well as rats 4 weeks following LAD ligation treated with vehicle (CHF, n = 12) or losartan (CHF+losaratan, n = 9). The * indicates a significant difference (p<0.05) compared to control.</p

The expression and activation of p38 MAPK does not change following LAD ligation.

<p>Western blots with anti-phospho-p38 MAPK antibody and anti-p38 MAPK antibody demonstrate that both p38 MAPK and phospho-p38 MAPK are barely detectable in aortic smooth muscle in control and rats following LAD ligation (CHF, CHF+losartan). Both p38 MAPK and phosphorylated p38 MAPK are detected in cultured rat SMCs. The Coomassie stained gel shows actin, the loading control.</p

There is a significant activation of p42/44 MAPK following LAD ligation, which is preserved with ARB.

<p>(A) Western blots demonstrate that while the expression of p42/44 MAPK does not change following LAD ligation, there is a significant increase in the phosphorylation (activation) of p42/44 MAPK following LAD ligation in the animals treated with vehicle (CHF), which returns to control levels with losartan therapy (CHF+losartan). In addition, treatment of control animals with losartan decreased the activation of p42/44 MAPK. The Coomassie stained gel shows actin, the loading control. (B) Bar graph represents the relative phosphorylation of p42/44 MAPK in control and animals after LAD ligation. The relative phosphorylation of p42/44 MAPK in the controls was set at 1, and the data represent mean±SEM for control (n = 9), and rats following LAD ligation treated with vehicle (CHF, n = 12) or losartan (CHF+losartan, n = 9), as well as control animal treated with losartan (Control+losartan, n = 4). The * indicates a significant difference compared to controls, while the ^∧ is a significant difference compared to losartan treatment.</p

Relative LZ+ MYPT1 expression falls following LAD ligation, which is preserved with ARB.

<p>(A) Western blots demonstrate that the relative expression of the LZ+ MYPT1 isoform falls following LAD ligation, but is preserved at control levels in the rats treated with losartan. The Coomassie stained gel shows actin, the loading control. (B) Bar graph represents the relative expression of the LZ+ MYPT1 isoform (given as LZ+ MYPT1/MYPT1) in control rats and animals after LAD ligation. The relative expression of LZ+ MYPT1 in the controls was set at 1. The data represent mean±SEM for control (n = 9), rats following LAD ligation treated with either vehicle (CHF, n = 12) or losartan (CHF+losartan, n = 9), and control animals treated with losartan (Control+losartan, n = 4). The * indicated a significant difference compared to controls, while the ^∧ is a significant difference compared to losartan treatment.</p

<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

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

<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

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