Loss of myocardial protection against myocardial infarction in middle-aged transgenic mice overexpressing cardiac thioredoxin-1

Abstract

Thioredoxin-1 (Trx1) protects the heart from ischemia/reperfusion (I/R) injury. Given that the age at which the first episode of coronary disease takes place has considerably decreased, life at middle-aged (MA) emerges as a new field of study. The aim was determine whether infarct size, Trx1 expression and activity, Akt and GSK-3β were altered in young (Y) and MA mice overexpressing cardiac Trx1, and in a dominant negative (DN-Trx1) mutant of Trx1. Langendorff-perfused hearts were subjected to 30 minutes of ischemia and 120 minutes of reperfusion (R). We used 3 and 12 month-old male of wild type (WT), Trx1, and DN-Trx1. Trx1 overexpression reduced infarct size in young mice (WT-Y: 46.8±4.1% vs. Trx1-Y: 27.6±3.5%, p < 0.05). Trx1 activity was reduced by 52.3±3.2% (p < 0.05) in Trx1-MA, accompanied by an increase in nitration by 17.5±0.9%, although Trx1 expression in transgenic mice was similar between young and middle-aged. The expression of p-Akt and p-GSK-3β increased during reperfusion in Trx1-Y. DN-Trx1 mice showed neither reduction in infarct size nor Akt and GSK-3β phosphorylation. Our data suggest that the lack of protection in Trx1 middle-aged mice even with normal Trx1 expression may be associated to decreased Trx1 activity, increased nitration and inhibition of p-Akt and p-GSK-3β.

Keywords: Gerotarget; ischemia/reperfusion; middle-aged; myocardial infarction; thioredoxin-1.

Figure 1. Infarct size expressed as a percentage of the total left ventricular area

Infarct size decreased significantly in Trx1 young group but this cardioprotective effect was abolished in Trx1 middle-aged. Panel B shows representative slices of the different experimental groups. Data are represented as mean +/− SEM. *: p < 0.05 vs. WT-Y. WT-Y: wild type in young animals (3 month-old); WT-MA: wild type in middle-aged animals (12 month-old); Trx1-Y: thioredoxin-1 young group; Trx1-MA: thioredoxin-1 middle-aged group.

Figure 2. Panel A shows an increased in Trx1 expression in transgenic animals, both in young (Y) and middle-aged (MA) group in normoxic (Nx) conditions and after ischemia/reperfusion protocol

The levels of Trx1 in wild type mice (WT) decreased at reperfusion (after 30 minutes of ischemia and 15 minutes of reperfusion) in young and middle-aged animals, but in transgenic mice the levels of Trx1 remains in similar values as normoxic conditions. Panel B show Ponceau S-stained blot, as loading control, and representative blots of Trx1. Panel C shows that in transgenic mice the activity was lower in Trx1 young group compared with Trx1 middle-aged group. Finally, in panel D we observed an increased in Trx1 nitration in middle-aged group compared with young group. Data are represented as mean +/− SEM. *: p < 0.05 vs. WT animals. #: p < 0.05 vs. Nx respective conditions; &: p < 0.05 vs. Trx1 young group.

Figure 3. Akt phosphorylation (Ser 473) protein expression in the cytosolic fraction of normoxic (Nx) and ischemia/reperfusion protocols (I/R) in young (Panel A) and middle-aged (Panel B) mice

There were not significantly changes in the cytosolic Akt protein expression (Panel C). Also, not significantly changes were detected, in the cytosolic p-Akt Ser 473 protein expression in Trx1 and WT middle-aged groups, however in Trx1 young group a significantly enhanced of Akt phosphorylation after I/R was detected. Panel C shows Ponceau S-stained blot, as loading control, and representative blots of total and phosphorylated Akt. Data are represented as mean +/− SEM. *: p < 0.05 vs normoxic Trx1; #: p < 0.05 vs WT-Y I/R. WT-Y: wild type in young animals (3 month-old); WT-MA: wild type in middle-aged animals (12 month-old); Trx1-Y: thioredoxin-1 young group; Trx1-MA: thioredoxin-1 middle-aged group.

Figure 4. pGSK-3β Ser 9 protein expression in the cytosolic fraction of normoxic (Nx) and ischemia/reperfusion protocols (I/R) in young (Panel A) and middle-aged (Panel B) mice

There were not significantly changes in the cytosolic GSK-3β protein expression (Panel C). Also, not significantly changes in the cytosolic in p-GSK-3β Ser 9 protein expression were detect in Trx1and WT middle-aged groups, however in Trx1 young group, a significantly enhanced of GSK-3β phosphorylation after I/R was detected. Panel C shows Ponceau S-stained blot, as loading control, and representative blots. Data are represented as mean +/− SEM. *:p < 0.05 vs normoxic Trx1; #: p < 0.05 vs WT-Y I/R. WT-Y: wild type in young animals (3 month-old); WT-MA: wild type in middle-aged animals (12 month-old); Trx1-Y: thioredoxin-1 young group; Trx1-MA: thioredoxin-1 middle-aged group.

Figure 5. We showed DN-Trx1 mice results in order to confirm that the cardioprotection conferred by Trx1 involves Akt and GSK-3β inhibition/phosphorylation

nfarct size (Panel A), Trx1 expression (Panel C), p-Akt Ser473 (Panel D) and p-GSK-3β Ser9 (Panel E) were similar between wild type (WT) and in dominant negative for Trx1 (DN-Trx1) in young and middle-aged mice. Panel B shows representative slide of infarct size. Panel F shows Ponceau S-stained blot, as loading control, and Akt/GSK-3β representative blots. Data are represented as mean +/− SEM.