PDE5 expression in cardiac tissue from mice with chronic pressure overload.

<p>(<b>A</b>) Immunoblot analysis showed increased PDE5 protein expression after chronic pressure overload. Protein levels of GAPDH were measured to control for sample variability. (<b>B</b>) Immunohistochemical staining of PDE5 demonstrated that increased PDE5 expression was present in cardiac myocytes. Colocalization was confirmed in confocal images of double immunofluorescent staining of PDE5 (green) and desmin (red). Scale bars, 20 µm.</p

Hemodynamic parameters in PDE5-TG and WT after 10 weeks TAC.

<p>HW/BW indicates heart to body weight ratio; RCA-LCA gradient, gradient between right and left common carotid artery; dP/dt_max and dP/dt_min, maximum and minimum of the first derivative of LV pressure over time; Tau, time constant for isovolumic relaxation (Weiss); and HR, heart rate. *<i>P</i><0.05 vs WT.</p

Ca²⁺-sensitivity of active force, and passive force in PDE5-TG and WT cardiac myocytes after chronic pressure overload.

<p>(<b>A</b>) In the Ca²⁺-force relationship, the Ca²⁺-sensitivity of isometric force production represents the [Ca²⁺] (plotted as a function of pCa; pCa = −log₁₀[Ca²⁺]) evoking half-maximal force production (<i>top panel</i>). Ca²⁺-sensitivity did not differ between PDE5-TG and WT cardiac myocytes, and was reduced after pretreatment with PKG (<i>bottom panel</i>). (<b>B</b>) Passive forces were significantly greater in cardiac myocytes from PDE5-TG compared to WT cardiac myocytes. Pretreatment of cardiac myocytes with PKG significantly reduced the passive forces in both genotypes. *<i>P</i><0.05; ^†<i>P</i><0.05 vs without PKG.</p

Indices of cardiac remodeling after 10 weeks TAC in WT mice with increased PDE5 expression.

<p>HW/BW indicates heart to body weight ratio; LVID_D, LV internal diameter during diastole; EDV, end-diastolic volume; LVID_S, LV internal diameter during systole; ESV, end-systolic volume; FS, fractional shortening; EF, ejection fraction; and HR, heart rate. <b>^†</b><i>P</i><0.05 vs baseline, ^*<i>P</i><0.05 vs mice with moderately increased PDE5 expression.</p

PDE5 expression in LV outflow tract tissue from patients with severe aortic stenosis.

<p>(<b>A</b>) In cardiac tissue of control subjects, PDE5 expression was limited. In contrast, in AS patients, marked PDE5 immunoreactivity was present in scattered cardiac myocytes. (<b>B</b>) Double immunofluorescent staining of PDE5 (green) and desmin (red) confirmed protein expression in cardiac myocytes. Scale bars, 25 µm.</p

Grußwort

<p>The flow-chart shows the experimental procedures performed at certain time-points during the study. GD, gestational day; PPD, postpartum day; PFU, plaque forming unit.</p

Myocardial levels of SERCA2 in PDE5-TG and WT after 10 weeks TAC.

<p>Transcript levels (<b>A</b>) and protein levels (<b>B</b>) of SERCA2, measured using RT-qPCR and immunoblot and densitometric analysis, respectively, were significantly lower in PDE5-TG than in WT. Transcript and protein levels of GAPDH were measured for normalization. ^*<i>P</i><0.05 vs WT.</p

<i>FLT1</i> protein isoforms and mRNA transcript variants.

<p>(A) Flt-1 contains seven extracellular Ig-like domains and an intracellular tyrosine kinase. The first three extracellular Ig-like domains are essential for ligand-binding, while the 4–7th extracellular Ig-like domains for receptor dimerization. The truncated mouse sFlt-1 mutant [msFlt-1(1–3)] contains only 1–329 amino acids of Flt-1, corresponding to the first three Ig-like domains. Mouse and human sFlt-1-i13 contains the first six Ig-like domains corresponding to 1–657 amino acids of Flt-1, as well as a unique 31-amino-acid tail. This unique C-terminus is evolutionarily highly conserved among mammals; the mouse and human amino acid sequences of this tail are only different in two positions (shown with blue letters). Among the human placental expressed sFlt-1 isoforms, hsFlt-1-i14, hsFlt-1-e15a and hsFlt-1-e15b diverge from Flt-1 after amino acid 706, and contain a 31-, 28- and 13-amino-acid unique tails, respectively. (B) Among the placental expressed <i>FLT1</i> transcripts, the abundance of the mRNA encoding for the transmembrane receptor is about 2.5% in preeclampsia. <i>FLT1</i> transcript expression data was retrieved from Jebbink et al. and is shown as transcript level divided by total <i>FLT1</i> transcript level [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119547#pone.0119547.ref154" target="_blank">154</a>]. HsFlt-1-i13, the second most abundant placental <i>FLT1</i> transcript in preeclampsia, is generated by skipped splicing and extension of exon 13. Similarly, hsFlt-1-i14 is generated by skipped splicing and extension of exon 14. HsFlt-1-e15a and hsFlt-1-e15b contain alternatively spliced exons derived from intronic sequences (exon 15a and exon 15b, respectively). The most abundant placental transcript, hsFlt-1-e15a contains exon 15a, which is located within a primate-specific AluSeq retrotransposon. The graph was adapted with permission from figures in publications of Heydarian et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119547#pone.0119547.ref153" target="_blank">153</a>] and Shibuya M. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119547#pone.0119547.ref243" target="_blank">243</a>]. Permissions for reuse of these original figures were obtained from Elsevier Ltd. and from the Proceedings of the Japan Academy, Series B, respectively.</p

“EM35” mouse had early-onset preeclampsia-like symptoms associated with fetal growth restriction.

<p>(A) “EM35” mouse had a higher and earlier blood pressure peak than other hsFlt-1-e15a-treated mice, followed by the normalization of blood pressures and a second blood pressure peak on PPD1. (B) The urinary albumin/creatinine ratio on GD18 was higher in “EM35” mouse than in other hsFlt-1-e15a-treated mice. (C) HsFlt-1-e15a expression in the liver was lower in “EM35” mouse than in other hsFlt-1-e15a-treated mice. (D-E) Histopathological examination of the liver found a multiple cystic biliary hyperplasia in the liver (* in D), and multiple recent (D) and remote (E) infarcts in the parenchyma of liver. H&E staining, 200x magnifications. (F) The endothelial outgrowth area was the lowest in “EM35” mouse among hsFlt-1-e15a-treated mice. (G-H) Mean fetal (G) weights (0.749±0.029) and placental (H) weights (0.080±0.006) in “EM35” mouse were significantly lower than in other hsFlt-1-e15a-treated mice (fetal weight: 1.06±0.023, p = 2.017x10^-15, placental weight: 0.111±0.004, p = 2.05x10^-6). (I) Histopathological examination of a placenta from “EM35” mouse showed multiple thrombi in maternal decidual vessels (star and black box). H&E staining, 20x magnification. The image inside the black box is magnified to 800x in Subfigure I. (J) The arrow shows one of the thrombi.</p

Profiling of hsFlt-1-e15a and GFP expression.

<p>Boxplots show the expression profile of GFP (A) or hsFlt-1-e15a (B) mRNAs in placentas harvested on GD18 and in five tissues harvested on PPD8. Adenovirus doses (1x10⁹ or 2x10⁹ plaque-forming units) are depicted with different colors. Expression of hsFlt-1-e15a and GFP mRNAs was highest in the liver. Viral dose-effect was not seen in GFP expression; however, there was a dose-effect in hsFlt-1-e15a expression. The number of hsFlt-1-e15a expressing placenta, brain and uterine tissues was low in the hsFlt-1-e15a 1x group.</p