Inhibition of PKCβ2 overexpression ameliorates myocardial ischaemia/reperfusion injury in diabetic rats via restoring caveolin-3/Akt signaling

Abstract

Activation of PKCβ (protein kinase Cβ) plays a critical role in myocardial I/R (ischaemia/reperfusion) injury in non-diabetic rodents. In the myocardium of diabetes, PKCβ2 overexpression is associated with increased vulnerability to post-ischaemic I/R injury with concomitantly impaired cardiomyocyte Cav (caveolin)-3 and Akt signalling compared with non-diabetic rats. We hypothesized that myocardial PKCβ overexpression in diabetes exacerbates myocardial I/R injury through impairing Cav-3/Akt signalling. Streptozotocin-induced diabetic rats were treated with the selective PKCβ inhibitor ruboxistaurin (RBX, 1 mg/kg per day) for 4 weeks, starting from 1 week after diabetes induction, before inducing myocardial I/R achieved by occluding the left descending coronary artery followed by reperfusion. Cardiac function was measured using a pressure-volume conductance system. In an in vitro study, cardiac H9C2 cells were exposed to high glucose (30 mmol/l) and subjected to hypoxia followed by reoxygenation (H/R) in the presence or absence of the selective PKCβ2 inhibitor CGP53353 (1 μmol/l), siRNAs of PKCβ2 or Cav-3 or Akt. Cell apoptosis and mitochondrial membrane potential were assessed by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) and JC-1 staining respectively. RBX significantly decreased post-ischaemic myocardial infarct size (35±5% compared with 49±3% in control, P<0.05) and attenuated cardiac dysfunction, and prevented the reduction in cardiac Cav-3 and enhanced phosphorylated/activated Akt (p-Akt) in diabetic rats (P<0.05). H/R increased cardiomyocyte injury under high glucose conditions as was evident by increased TUNEL-positive and increased JC-1 monomeric cells (P<0.05 compared with control), accompanied with increased PKCβ2 phosphorylation/activation and decreased Cav-3 expression. Either CGP53353 or PKCβ2 siRNA significantly attenuated all of these changes and enhanced p-Akt. Cav-3 gene knockdown significantly reduced p-Akt and increased post-hypoxic cellular and mitochondrial injury despite a concomitant reduction in PKCβ2 phosphorylation. PKCβ2 inhibition with RBX protects diabetic hearts from myocardial I/R injury through Cav-3-dependent activation of Akt.

Repeated Non-Invasive Limb Ischemic Preconditioning Confers Cardioprotection Through PKC-Ԑ/STAT3 Signaling in Diabetic Rats

“…MMP loss was measured using a JC-1 mitochondrial membrane potential kit (Cayman Chemical) as described [31]. JC-1 is a lipophilic, cationic dye and its advantage is that it can selectively enter into mitochondria based on the aggregation of the probe and reversibly change color from green to red as the membrane potential increases.…”

Propofol Through Upregulating Caveolin-3 Attenuates Post-Hypoxic Mitochondrial Damage and Cell Death in H9C2 Cardiomyocytes During Hyperglycemia

“…Sample cells were collected and washed twice with PBS; the cells were collected through centrifugation, then resuspended in 500 µL JC-1 staining solution and incubated for 15 min in an incubator with 5% CO 2 under 37 °C; the cells were collected again through centrifugation, and then resuspended in 500 µL preheated incubation buffer. The results were detected and analyzed by a flow cytometer as we described [21]. …”

“…Interestingly, in airway smooth muscle cells, propofol mediated inhibition of cellular contracture was diminished in cells with Cav-1 gene knock-down [18], suggesting that Cav may play critical roles in propofol beneficial effects. However, we and others have found that cardiac Cav-3 is reduced in hearts from diabetic animals [19, 20] and that this reduction was associated with increased mitochondrial dysfunction [21] and exacerbated myocardial I/R injury [19, 22]. It is unknown whether or not Cav-3 plays a role in propofol cardioprotection in hearts from diabetes.…”

AMPK Contributes to Cardioprotective Effects of Pterostilbene Against Myocardial Ischemia- Reperfusion Injury in Diabetic Rats by Suppressing Cardiac Oxidative Stress and Apoptosis

“…Four weeks after PT treatment, myocardial IR (30 minutes ischemia/2 hours reperfusion) was employed [32]. There were four groups (n=8/group), namely, 1) D sham group (receiving vehicle); 2) D + IR group (receiving vehicle); 3) D + IR + Pterostilbene 20 mg/kg/d (PT 20) group; 4) D + IR + Pterostilbene 40 mg/kg/d (PT 40) group.…”