Effects of acute n-3 TG emulsion treatment on apoptosis and autophagy proteins in H/R.

<p>(A) Western blot analysis of Bcl-2 and Beclin-1 in hearts subjected to I/R injury with or without n-3 TG emulsion administered during 1h reperfusion. Bar graphics show (B) Bcl-2 and (C) Beclin-1 protein expression in I/R vs n-3 TG treated hearts. n = 3–4 mice hearts/group. Data represent means ± SD</p

Effects of acute n-3 TG emulsion treatment on signaling pathways in H/R.

<p>(A) Western blot analysis of p-AKT and p-GSK3β in hearts subjected to I/R injury with or without n-3 TG emulsion administrated during 1h reperfusion. Bar graphs show (B) p-AKT/t-AKT and (C) p-GSK3β/t-GSK3β protein expression in I/R vs I/R n-3 TG treated hearts. n = 3–4 mice hearts/group. Data represent means ± SD.</p

Effects of acute n-3 TG emulsion injection in <i>in vivo</i> LAD occlusion model.

<p>Mice were subjected to LAD occlusion for 30min followed by reperfusion (48h) with or without acute n-3 TG emulsion injections. Hearts were retrieved at 48h post-LAD ligation and subjected to TTC staining. (A) The analysis of infarct area and area-at-risk (AAR) in the myocardium were determined in I/R vs I/R n-3 TG groups. n = 5–6 mice/group. AAR was no-significant (ns) (B) Plasma collected at 48h was analyzed for total LDH levels in I/R vs I/R n-3 TG groups. n = 5–6 mice/group. (C) Measurements of cardiac function using echocardiography were performed at 48h post-LAD ligation. Changes in % fractional shortening (FS) are reported for each group. n = 5–6 mice/group. Data represent means ± SD.</p

Effects of acute n-3 TG emulsion treatment on PPARγ protein expression.

<p>(A) Western blot analysis of PPARγ in hearts subjected to I/R injury with or without n-3 TG emulsion administered during 1h reperfusion. (B) Bar graph shows PPARγ protein expression. n = 3–4 mice hearts/group. Data represent means ± SD.</p

Aldose reductase modulates acute activation of mesenchymal markers via the β-catenin pathway during cardiac ischemia-reperfusion

<div><p>Aldose reductase (AR: human, AKR1B1; mouse, AKR1B3), the first enzyme in the polyol pathway, plays a key role in mediating myocardial ischemia/reperfusion (I/R) injury. In earlier studies, using transgenic mice broadly expressing human <i>AKR1B1</i> to human-relevant levels, mice devoid of <i>Akr1b3</i>, and pharmacological inhibitors of AR, we demonstrated that AR is an important component of myocardial I/R injury and that inhibition of this enzyme protects the heart from I/R injury. In this study, our objective was to investigate if AR modulates the β-catenin pathway and consequent activation of mesenchymal markers during I/R in the heart. To test this premise, we used two different experimental models: <i>in viv</i>o, <i>Akr1b3</i> null mice and wild type C57BL/6 mice (WT) were exposed to acute occlusion of the left anterior descending coronary artery (LAD) followed by recovery for 48 hours or 28 days, and <i>ex-vivo</i>, WT and <i>Akr1b3</i> null murine hearts were perfused using the Langendorff technique (LT) and subjected to 30 min of global (zero-flow) ischemia followed by 60 min of reperfusion. Our <i>in vivo</i> results reveal reduced infarct size and improved functional recovery at 48 hours in mice devoid <i>of Akr1b3</i> compared to WT mice. We demonstrate that the cardioprotection observed in <i>Akr1b3</i> null mice was linked to acute activation of the β-catenin pathway and consequent activation of mesenchymal markers and genes linked to fibrotic remodeling. The increased activity of the β-catenin pathway at 48 hours of recovery post-LAD was not observed at 28 days post-infarction, thus indicating that the observed increase in β-catenin activity was transient in the mice hearts devoid of <i>Akr1b3</i>. In <i>ex vivo</i> studies, inhibition of β-catenin blocked the cardioprotection observed in <i>Akr1b3</i> null mice hearts. Taken together, these data indicate that AR suppresses acute activation of β-catenin and, thereby, blocks consequent induction of mesenchymal markers during early reperfusion after myocardial ischemia. Inhibition of AR might provide a therapeutic opportunity to optimize cardiac remodeling after I/R injury.</p></div

β-Catenin pathway inhibitor reverses the <i>Akr1b3</i> null mediated protection and mesenchymal marker expression.

<p>Mice hearts were subjected to <i>ex vivo</i> I/R using Langendorff technique. (A) qRT-PCR on heart tissues for mesenchymal markers as indicated, N = 3. (B) qRT-PCR on heart tissues for collagen and matrix metalloproteinases as indicated, N = 3 mice/group. (C) qRT-PCR on primary CF for mesenchymal markers treated either with recombinant TGFB2 or IWR as indicated, N = 5/group. Error bars represent mean ± SEM. * p<0.05, unless otherwise noted.</p

Increased expression of fibrotic factors in <i>Akr1b3</i> null I/R mice hearts.

<p>RNA isolated from heart tissues from mice subjected to I/R. (A) qRT-PCR on collagens (<i>Col1a1</i>, <i>Col1a2</i>, <i>Col3a1)</i> and MMPs (<i>Mmp2</i> and <i>Mmp9)</i>, N = 5 mice/group. (B) Western blot analysis on RUNX2 and MMP2 expression in heart tissues subjected to I/R. N = 4/group. Error bars represent mean ± SEM. * p<0.05, unless otherwise noted.</p

Mesenchymal activation in <i>Akr1b3</i> null mice hearts 48 h post-I/R.

<p>RNA isolated from heart tissues from mice subjected to I/R. (A) qRT-PCR on EndoMT transcription factors- <i>Snai1</i> and <i>Snai2</i>. (B) qRT-PCR on mesenchymal markers as indicated. (C) qRT-PCR on endothelial markers as indicated. MAECs transfected with scrambled and siR against <i>Akr1b3</i> and probed for (D) <i>Akr1b3</i>. € qRT-PCR on EndoMT transcription factors as indicated. (F) qRT-PCR on mesenchymal markers as indicated. (G) qRT-PCR on endothelial markers as indicated. N = 3 mice/group. Error bars represent mean ± SEM. * p<0.05, unless otherwise noted.</p

Cardioprotection in <i>Akr1b3</i> null I/R mice.

<p>Male WT and <i>Akr1b3</i> null mice were subjected to LAD occlusion followed by reperfusion at age 4 months. (A) Western blot analysis of AKR1B3 in heart tissue lysate at 48 h post-LAD was performed and normalized to levels of B-ACTIN, N = 4 mice/genotype. (B) <i>Akr1b3</i> null mice exhibit decreased infarct area (expressed as in % of infarct area/area at risk) after LAD/reperfusion vs. WT mice (n = 10/group; * p<0.05 vs. WT LAD) with no genotype differences in area at risk (data not shown). (C) Total plasma LDH levels were measured at 48 h post-LAD, N = 6 mice/group. (D) Changes in % fractional shortening (FS) with representative echocardiographic image. (E) % of fractional area change (FAC), N = 10/group. (F) The ratio of heart weight to body weight was measured, N = 10/group. Error bars represent mean ± SEM. * p<0.05, unless otherwise noted.</p

Effect of n-3 TG emulsion <i>on in vitro</i> I/R model.

<p>(A) Determination of myocardial ischemic injury was assessed by left ventricular developed pressure (LVDP) recovery in hearts subjected to I/R and treated with or without n-3 TG emulsion during reperfusion. (B) Heart perfusates were collected after 1h reperfusion to detect total LDH levels. n = 4–5 mice/group. Data represent means ± SD.</p