Acute myocardial infarction, the clinical manifestation of ischemia-reperfusion (IR) injury, is a leading cause of death worldwide. Although percutaneous coronary interventions and thrombolytic therapies are effective in limiting the duration of ischemia, the re-introduction of blood flow to previously ischemic area causes additional damage, collectively known as reperfusion injury. One of the most effective ways to reduce reperfusion injury is ischemic preconditioning (IPC), which is induced by several cycles of brief ischemia and reperfusion bouts prior to the prolonged ischemia. IPC was found to be mediated through signaling pathways (including activation of Src, PI3K-IB, and PKCe), and mimicked by a number of pharmacological or mechanical interventions. However, 25 years after the first report of IPC, preconditioning research has not translated into clinical application against cardiac reperfusion injury. Contributing to this somewhat surprising and disappointing failure to translate preconditioning into the clinic, the applicability and efficacy of preconditioning treatments in the setting of acute myocardial infarction (MI) have not always been carefully considered in the research setting. In particular, the impact of comorbidities on cardioprotective signaling or the unpredictable nature of MI has limited the impact of IPC. Against this background, the overall objective of this work was to investigate the potential benefit of using cardiac glycosides (CG) drugs to trigger cardioprotection in conditions relevant to acute myocardial infarction.
Indeed, treatment with low doses of the CG ouabain before ischemia has been shown to induce cardioprotective effects against IR injury through a mechanism known as ouabain preconditioning (OPC). Rather than the classic specific inhibition of Na/K-ATPase-mediated ion transport, the mechanism underlying OPC is the activation of the more recently recognized signaling function of Na/K-ATPase, which includes Src-PKCe, ROS and the mKATP channel.
Accordingly, a first study was undertaken to test whether digoxin, which is the only FDA-approved CG, could mimic OPC. Further, we tested whether CG could improve cardiac recovery when administered at the onset of reperfusion rather than before ischemia (i.e. postconditioning rather than preconditioning), which is highly suitable in the setting of acute MI. In Langendorff-perfused mouse heart preparations, ouabain and digoxin 10 µM similarly inhibited Na/K-ATPase activity by about 30% and activated PKCe translocation by about 50%. Four min of perfusion with 10 µM ouabain (OPC protocol) or digoxin (DigPC protocol), followed by 8 min washout period prior to 40 min ischemia and 30 min reperfusion significantly improved the recovery of left ventricle developed pressure (LVDP), end-diastolic pressure (EDP), and protected Na/K-ATPase activity. Ouabain and digoxin treatments also significantly decreased LDH release and reduced infarct size, in a similar manner, further suggesting protection against IR-induced cell death. These findings were consistent with previous reports of OPC-induced protection against IR in the rat and rabbit cardiac tissue, and revealed that OPC and DigPC were equally effective. Postconditioning protocols consisting of a single bolus injection of 100 nmoles of ouabain or digoxin in the coronary tree at the beginning of reperfusion revealed that both ouabain and digoxin postconditioning improved the recovery of LVDP and decreased LDH release, suggesting a functional and structural protection as efficient as the one provided by OPC. This series of experiments therefore led to the conclusion that both CG ouabain and FDA-approved digoxin are effective preconditioners and postconditioners. Digoxin postconditioning protocols may be of particular interest in clinical settings.
Our second study explored mechanistic differences between IPC and OPC, which could make the CG-based interventions more suitable in diseases where IPC has reduced efficiency. Specifically, 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. 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 10µM and IPC 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 Ser473 phosphorylation. The PI3K-IA inhibitor PI-103 (100 nM) blocked ouabain-induced Akt activation. IPC and OPC significantly preserved cardiac contractile function and tissue viability as evidenced by left ventricular developing pressure and end-diastolic pressure recovery, reduced lactate dehydrogenase release, and decreased infarct size. OPC protection was blunted by PI-103 but not by the PI3K-IB inhibitor AS-604850 (1 µM). In contrast, IPC-mediated protection was not affected by PI-103 but was blocked by AS-604850 (15 µM). These data suggested that PI3K-IA activation is required for OPC while PI3K-IB activation is needed for IPC. Ouabain-induced PKCe translocation, previously shown to be critical for OPC, was unaffected by PI-103 co-treatment and further studies shall reveal the identity of the downstream targets of this new PI3K IA-dependent branch of OPC.
In summary, cardiac glycosides can work in both preconditioning and postconditioning manners. In addition, cardiac glycosides preconditioning is mediated through PI3K-IA while ischemic preconditioning (IPC) relies on PI3K-IB activation. These findings may be of clinical relevance in the setting of AMI in patients presenting with diseases and medications that could differentially affect the integrity of cardiac PI3K-IA and IB pathways.