SERCA activity, PLB phosphorylation and SERCA protein expression levels were measured.

<p>Using the same tissue obtained from each group as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048115#pone-0048115-g002" target="_blank">figure 2</a>, SERCA activity (Panel A), phosphorylation level of PLB (representative protein bands shown in Panel B and quantification in bar graph of Panel C) and SERCA protein expression level (Panel D & E) were quantified also shown. All data expressed as mean±SD. * denotes P<0.05 vs. Sham groups at the same time point and †, P<0.05 vs. I/R group at the same time point.</p

The role of PI3K/Akt signaling in the protection by fasudil was evaluated by using an Akt inhibitor, LY294002.

<p>Sham group, I/R group and I/R+fasudil group were either given LY294002 or placebo (n = 6 for each group rats treated with LY294002 for both time points, all the other rats are the same as shown in above). Western blotting was performed, targeting phosphorylation levels of Akt (representative protein bands shown in Panel A and quantified in bar graph in Panel B); JAK2 (Panel C & D), STAT3 (Panel E & F) and PLB (Panel G & H). SERCA protein expression levels (Panel I & J) were also analyzed. All data expressed as mean±SD. * denotes P<0.05 vs. Sham groups; †, P<0.05 vs. I/R group at the same time point and ‡, P<0.05 vs. I/R+fasudil group at the same time point.</p

Fasudil Protects the Heart against Ischemia-Reperfusion Injury by Attenuating Endoplasmic Reticulum Stress and Modulating SERCA Activity: The Differential Role for PI3K/Akt and JAK2/STAT3 Signaling Pathways

<div><p>Disordered calcium homeostasis can lead to endoplasmic reticulum (ER) stress. Our previous data showed that time course activation of ER stress contributes to time-related increase in ischemia-reperfusion (I/R) injury. However, it has not been tested whether PI3K/Akt and JAK2/STAT3 pathways play differential roles in reducing ER stress to protect the heart. In the present study, using fasudil which is a specific inhibitor of ROCK, we aimed to investigate whether improved SERCA expression and activity accounts for reduced ER stress by ROCK inhibition, specifically whether PI3K/Akt and JAK2/STAT3 pathways are differentially involved in modulating SERCA activity to reduce ER stress and hence I/R injury. The results showed that during the reperfusion period following 45 min of coronary ligation the infarct size (IS) increased from 3 h of reperfusion (45.4±5.57%) to 24 h reperfusion (64.21±5.43, P<0.05), which was associated with ER stress dependent apoptosis signaling activation including CHOP, Caspase-12 and JNK (P<0.05, respectively).The dynamic ER stress activation was also related to impaired SERCA activity at 24 h of reperfusion. Administration of fasudil at 10 mg/Kg significantly attenuated ROCK activation during reperfusion and resulted in an improved SERCA activity which was closely associated with decreases in temporal activation of ER stress and IS changes. Interestingly, while both PI3K/Akt and JAK2/STAT3 signaling pathways played equal role in the protection offered by ROCK inhibition at 3 h of reperfusion, the rescued SERCA expression and activity at 24 h of reperfusion by fasudil was mainly due to JAK2/STAT3 activation, in which PI3K/Akt signaling shared much less roles.</p> </div

Different SERCA isoform expressions were quantified.

<p>Using the same heart tissue described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048115#pone-0048115-g002" target="_blank">figure 2</a>, western blotting was performed using specific antibodies to measure the expression levels of different isoforms of SERCA. Panel A showed representative bands of SERCA2a for each group rats with quantification shown in bar graph, Panel B showed quantification of SERCA2b, and Panel C for SERCA3. All data expressed as mean±SD. * denotes P<0.05 vs. Sham groups; †, P<0.05 vs. I/R group at the same time point.</p

Time course increases in I/R injury associated with dynamic changes in ER stress signalings.

<p>In panel A, ER stress related protein markers including GRP78, phosphorylation level of JNK, cleaved Caspase-12 band and CHOP were quantified for IR rats in comparison with Sham operated rats. Apoptosis signaling was assessed by TUNEL staining using paraffin embedded heart tissue (Panel B) with bar graph showing percentage TUNEL staining positive cells (Panel C). Caspase-3 activity was also quantified (Panel D) (n = 6 for I/R group and n = 4 for sham group at each time point). Representative sequential LV slices obtained from each group in panel E show both area at risk demarcated with Evans blue staining for normal heart tissue and area of necrosis with 2,3,5-triphenyltetrazolium chloride staining (pale area = infarcted tissue). The bar graph shows the percentage of LV weight of infarct area over LV weight at area at risk (Panel F), demonstrating time course changes in infarct size at 3, 12 and 24 hours of reperfusion following 45 minutes ischemia (n = 8 for each time point/group). All the data presented in the bar graphs were expressed as mean±SD. * denotes P<0.05 vs. Sham groups; †, P<0.05 vs. I/R group at 3 hours of reperfusion and ‡, P<0.05 vs. I/R group at 12 hours of reperfusion.</p

ROCK activity was measured in the presence of either PI3K/Akt inhibitor, LY294002, or JAK2 inhibitor, AG490, by quantifying the phosphorylation level of ERM.

<p>Representative bands for p-ERM and total ERM were shown for each group rats (n = 6 for either sham or I/R group rats treated with LY294001 or AG490, the other group rats are the same as shown above). All data expressed as mean±SD. * denotes P<0.05 vs. sham group.</p

The role for JAK2/STAT3 signaling pathway was also tested by using pharmacological inhibitor.

<p>Sham group, I/R group and I/R+fasudil group were either given AG490 or placebo (n = 6 for each group rats treated with AG490 for both time points, all the other rats are the same as shown in above). Western blotting was performed, targeting phosphorylation levels of Akt (Panel A showed representative protein bands for each group with quantification shown in bar graph in Panel B), JAK2 (Panel C & D), STAT3 (Panel E & F) and PLB (Panel G & H). SERCA protein expression levels (Panel I & J) were also analyzed. All data expressed as mean±SD. * denotes P<0.05 vs. Sham groups at the same time point; †, P<0.05 vs. I/R group at the same time point and ‡, P<0.05 vs. I/R+fasudil group at the same time point.</p

Both PI3K/Akt and JAK2/STAT3 pathway activities were quantified.

<p>Using the same heart tissue described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048115#pone-0048115-g002" target="_blank">figure 2</a>, western blotting was performed for each group targeting phosphorylation levels of Akt (representative protein bands shown in Panel A and quantified in bar graph in Panel B), phosphorylation levels of JAK2 (Panel C and D) and STAT3 (Panel E and F). All data expressed as mean±SD. * denotes P<0.05 vs. Sham groups; †, P<0.05 vs. I/R group at the same time point and #, P<0.05 vs. I/R+fasudil group at 3 hours of reperfusion.</p

To roles of PI3K/Akt and JAK2/STAT3 signaling in ER stress modulated by fasudil were also evaluated.

<p>Using the same heart tissue obtained as described as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048115#pone-0048115-g005" target="_blank">Fig. 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048115#pone-0048115-g006" target="_blank">Fig. 6</a>, ER stress related apoptosis protein markers, including GRP78, phospho-JNK, cleaved Caspase-12 and CHOP were quantified (panel A). IS were measured for I/R rats receiving fasudil with either LY294002 or AG490 at both 3 and 24 hours (n = 8 at each time point per group), the results were compared with I/R group and I/R with fasudil group (using the same data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048115#pone-0048115-g002" target="_blank">figure 2</a>). Representative heart slices with TTC staining are shown in panel B with quantification shown in bar graph (Panel C). All data shown are expressed as mean±SD. * denotes P<0.05 vs. sham group at the same time point; †, P<0.05 vs. I/R group at the same time point; ‡, P<0.05 vs. I/R+fasudil group at the same time point and #, P<0.05, vs. I/R+fasudil+LY294002 group at 24 hours of reperfusion.</p

ROCK inhibition results in decreases in both I/R injury and ER stress activation.

<p>Representative bands of phosphorylated and total ERM (Panel A) with quantification shown in bar graph (Panel B) demonstrating that ERM was activated during I/R injury process and can be suppressed by fasudil infusion (n = 4 for sham operated and 6 for I/R group rats with or without fasudil therapy for each time point). IS was measured for I/R rats receiving placebo or fasudil therapy at both 3 and 24 hours (n = 8 for each group at each time point) after reperfusion with representative sequential heart slices shown in Panel C and IS quantification shown in bar graph (Panel D). Using the same rat heart samples as shown in Panel A, paraffin embedded heart tissue slices from LV papillary muscle level, TUNEL staining (Panel E) was perform with percentage of TUNEL staining positive cells quantification shown in bar graph (Panel F). Using heart tissue from risk area Caspase-3 activity (Panel G) was measured to quantify apoptosis. ER stress related apoptosis signaling was also quantified by measuring its protein markers including GRP78, p-JNK, cleaved Caspase-12 and CHOP, with representative bands quantified in corresponding bar graph (Panel H). All data expressed as mean±SD. * denotes P<0.05 vs. Sham groups; †, P<0.05 vs. I/R group at the same time point; ‡, P<0.05 vs. I/R group at 3 hours of reperfusion and #, P<0.05 vs. I/R+fasudil group at 3 hours of reperfusion.</p