Preconditioning and Postconditioning by Cardiac Glycosides in the Mouse Heart.

Ouabain preconditioning (OPC) initiated by low concentrations of the cardiac glycoside (CG) ouabain binding to Na/K-ATPase is relayed by a unique intracellular signaling and protects cardiac myocytes against ischemia/reperfusion injury. To explore more clinically applicable protocols based on CG properties, we tested whether the FDA-approved CG digoxin could trigger cardioprotective effects comparable with those of ouabain using PC, preconditioning and PostC, postconditioning protocols in the Langendorff-perfused mouse heart subjected to global ischemia and reperfusion. Ouabain or digoxin at 10 µmol/L inhibited Na/K-ATPase activity by approximately 30% and activated PKCε translocation by approximately 50%. Digoxin-induced PC (DigPC), initiated by a transient exposure before 40 minutes of ischemia, was as effective as OPC as suggested by the recovery of left ventricular developed pressure, end-diastolic pressure, and cardiac Na/K-ATPase activity after 30 minutes of reperfusion. DigPC also significantly decreased lactate dehydrogenase release and reduced infarct size, comparable with OPC. PostC protocols consisting of a single bolus injection of 100 nmoles of ouabain or digoxin in the coronary tree at the beginning of reperfusion both improved significantly the recovery of left ventricular developed pressure and decreased lactate dehydrogenase release, demonstrating a functional and structural protection comparable with the one provided by OPC. Given the unique signaling triggered by OPC, these results suggest that DigPostC could be considered for patients with risk factors and/or concurrent treatments that may limit effectiveness of ischemic PostC.

Ischemia/reperfusion-induced alterations of enzymatic and signaling functions of the rat cardiac Na+/K+-ATPase: protection by ouabain preconditioning.

Cardiac glycosides (CG) are traditionally known as positive cardiac inotropes that inhibit Na+/K+-ATPase-dependent ion transport. CG also trigger-specific signaling pathways through the cardiac Na+/K+-ATPase, with beneficial effects in ischemia/reperfusion (I/R) injury (e.g., ouabain preconditioning, known as OPC) and hypertrophy. Our current understanding of hypersensitivity to CG and subsequent toxicity in the ischemic heart is mostly based on specific I/R-induced alterations of the Na+/K+-ATPase enzymatic function and has remained incomplete. The primary goal of this study was to investigate and compare the impact of I/R on Na+/K+-ATPase enzymatic and signaling functions. Second, we assessed the impact of OPC on both functions. Langendorff-perfused rat hearts were exposed to 30 min of ischemia and 30 min of reperfusion. At the inotropic concentration of 50 µmol/L, ouabain increased ERK and Akt phosphorylation in control hearts. In I/R hearts, this concentration did not induced positive inotropy and failed to induce Akt or ERK phosphorylation. The inotropic response to dobutamine as well as insulin signaling persisted, suggesting specific alterations of Na+/K+-ATPase. Indeed, Na+/K+-ATPase protein expression was intact, but the enzyme activity was decreased by 60% and the enzymatic function of the isoform with high affinity for ouabain was abolished following I/R. Strikingly, OPC prevented all I/R-induced alterations of the receptor. Further studies are needed to reveal the respective roles of I/R-induced modulations of Na+/K+-ATPase enzymatic and signaling functions in cardiomyocyte death.

Role of phosphoinositide 3-kinase IA (PI3K-IA) activation in cardioprotection induced by ouabain preconditioning.

Acute myocardial infarction, the clinical manifestation of ischemia-reperfusion (IR) injury, is a leading cause of death worldwide. Like ischemic preconditioning (IPC) induced by brief episodes of ischemia and reperfusion, ouabain preconditioning (OPC) mediated by Na/K-ATPase signaling protects the heart against IR injury. Class I PI3K activation is required for IPC, but its role in OPC has not been investigated. While PI3K-IB is critical to IPC, studies have suggested that ouabain signaling is PI3K-IA-specific. Hence, a pharmacological approach was used to test the hypothesis that OPC and IPC rely on distinct PI3K-I isoforms. In Langendorff-perfused mouse hearts, OPC was initiated by 4 min of ouabain 10 µM and IPC was triggered by 4 cycles of 5 min ischemia and reperfusion prior to 40 min of global ischemia and 30 min of reperfusion. Without affecting PI3K-IB, ouabain doubled PI3K-IA activity and Akt phosphorylation at Ser(473). IPC and OPC significantly preserved cardiac contractile function and tissue viability as evidenced by left ventricular developed pressure and end-diastolic pressure recovery, reduced lactate dehydrogenase release, and decreased infarct size. OPC protection was blunted by the PI3K-IA inhibitor PI-103, but not by the PI3K-IB inhibitor AS-604850. In contrast, IPC-mediated protection was not affected by PI-103 but was blocked by AS-604850, suggesting that PI3K-IA activation is required for OPC while PI3K-IB activation is needed for IPC. Mechanistically, PI3K-IA activity is required for ouabain-induced Akt activation but not PKCε translocation. However, in contrast to PKCε translocation which is critical to protection, Akt activity was not required for OPC. Further studies shall reveal the identity of the downstream targets of this new PI3K IA-dependent branch of OPC. These findings may be of clinical relevance in patients at risk for myocardial infarction with underlying diseases and/or medication that could differentially affect the integrity of cardiac PI3K-IA and IB pathways.