Gsk-3β Inhibitors Mimic the Cardioprotection Mediated by Ischemic Pre- and Postconditioning in Hypertensive Rats

The aim of this study was to examine the effects of GSK-3β inhibitors compared with PRE and POS in spontaneously hypertensive rats (SHR). Isolated hearts were submitted to the following protocols: IC: 45 min global ischemia (GI) and 1-hour reperfusion (R); PRE: a cycle of 5 min GI and 10 minutes of R prior to 45 min GI; POS: three cycles of 30 sec GI/30 sec R at the start of R. Other hearts received lithium chloride (LiCl) or indirubin-3′-monoxime,5-iodo-(IMI) as GSK-3β inhibitors. All interventions reduced the infarct size observed in IC group. The expressions of P-GSK-3β and P-Akt decreased in IC and were restored after PRE, POS, and GSK-3β inhibitors treatments. An increase of cytosolic MnSOD activity and lipid peroxidation and a decrease of GSH content observed in IC hearts were attenuated in PRE, POS, and LiCl or IMI treatments. An increase of P-GSK-3β/VDAC physical association and a partial recovery of mitochondrial permeability were also detected after interventions. These data show that, in SHR hearts, GSK-3β inhibitors mimic the cardioprotection afforded by PRE and POS and suggest that a decrease in mitochondrial permeability mediated by P-GSK-3β/VDAC interaction is a crucial event.

ventricular pressure (LVP) and CPP data were acquired by using an analog-to-digital converter and acquisition software (Chart V4.2.3 ADInstruments).

Infarct size determination
Infarct size was assessed by the widely validated triphenyltetrazolium chloride (TTC) staining technique [3]. At the end of reperfusion, atrial and right ventricular tissues were excised and left ventricle (VI) was frozen. The freeze VI was cut into six transverse slices, which were incubated for 5 minutes at 37°C in a 1% solution of triphenyltetrazolium chloride (TTC). To measure myocardial infarction, the slices were weighed and scanned.

Reduced glutathione (GSH)
GSH content was determined using the Ellman´s reagent. This method was based on the reaction of GSH with 5,5′ dithiobis (2-nitrobenzoic acid) to give a compound that absorbs at 412 nm. GSH levels were expressed as µg/mg of protein.

MnSOD cytosolic activity
SOD activity was measured by means of the nitroblue tetrazolium (NBT) method. Briefly, the supernatant was added to the reaction mixture of NBT with xanthine-oxidase, and the SOD activity measured colorimetrically in the form of inhibitory activity toward blue formazan formation by SOD in the reaction mixture. For measuring MnSOD activity, 5 mmol/l KCN was added to inhibit Cu-ZnSOD activity.

2.5.Immunoblotting
Other portion of LV was homogenized and mitochondrial and cytosolic fractions were GAPDH and VDAC/porin signals were used as a loading control of cytosolic and mitochondrial fractions, respectively.

Protein determination
The protein concentration was evaluated by the Bradford method [5] using bovine serum albumin as a standard.

Isolation of rat heart mitochondria.
LV were washed and homogenized in ice-cold isolation solution (IS) consisting of 75 mM sucrose, 225 mM mannitol, and 0.01 mM EGTA neutralized with Trizma buffer at pH 7.4.
After the tissue pieces were settled, the entire supernatant was discarded and fresh IS (5 ml) was added, and the mixture was transferred to a hand homogenizer. Proteinase (0.8 mg, bacterial, type XXIV, Sigma, formerly called Nagarse) was added just before starting the homogenization procedure. The whole homogenization procedure took no longer than 14 min in two steps of 7 min each (with 5 ml addition of fresh IS each). The homogenate was carefully transferred after each step to a polycarbonate centrifuge tube. After 5 min of 480×g of centrifugation to discard unbroken tissue and debris, the supernatant was centrifuged at 7 700×g for 10 min to sediment the mitochondria. The mitochondrial pellet was washed twice with IS and the last one with suspension solution (IS without EGTA) at 7,700×g for 5 min each.

Mitochondrial swelling Ca 2+ -induced
The ability of mitochondria to resist swelling was assessed by incubating 0.3 mg/mL of isolated mitochondria in a buffer containing (in mmol/L): 120 KCl, 20 MOPS, 10 Tris HCl, and 5 KH 2 PO4 adjusted to pH = 7.4. After 5-min preincubation, the mitochondria energized with the addition of 5 mmol/L succinate were induced to swell with 200 µmol/L CaCl 2 . If mPTP is open in the presence of Ca 2+ loading, solutes will be free to enter the inner matrix, causing the mitochondria to swell. These changes are observed as decreases of light scattering and followed using a temperature-controlled Hitachi F4500 spectrofluorometer operating with continuous stirring at excitation and emission wavelengths of 520 nm [6]. Light scattering decrease (LSD) was calculated for each sample by taking the difference of scattered light between before and after the addition of CaCl 2 .