The role of thioredoxin interacting protein and repressor activator protein 1 in myocardial ischemia reperfusion injury

Abstract

Myocardial ischemia reperfusion injury (IRI), following the recovery of arterial blood flow to the infarcted heart, is always an important issue in the treatment of myocardial infarction. The exact mechanisms haven’t been fully clarified and the development of effective treatment has been quite challenging, particularly for diabetic patients who tend to have worse myocardial IRI. Thioredoxin interacting protein (TXNIP), highly induced under hyperglycemia, is a pro-inflammatory protein that is strongly associated with oxidative stress. This thesis investigated the role of TXNIP to explain the development and exacerbation of myocardial IRI, especially in hyperglycemic status. Also, this thesis measured the protective effect of repeated non-invasive limb ischemic preconditioning (rNLIP) and analyzed its association with regulating TXNIP when treating diabetic myocardial IRI. Lastly, potential contribution to myocardial IRI pathogenesis by repressor activator protein 1 (Rap1) was explored.

The first study tested the cardio-protective effects of inhibiting TXNIP in treating hypoxia and reoxygenation (H/R) injury on H9C2 cardiomyocytes. The expression of TXNIP significantly increased after high glucose culture and also after H/R treatment. TXNIP knockdown by small interfering RNA (siRNA) transfection significantly improved cell viability, with lower lactate dehydrogenase (LDH) levels, and alleviated cell apoptosis after H/R in both normoglycemia and hyperglycemia. Besides, TXNIP knockdown saw remarkably less increase in the phosphorylation of IκBα and NF-κB together with down-regulated pro-inflammatory cytokines after H/R even with hyperglycemia. TXNIP knockdown also suppressed the elevation of NLR family pyrin domain containing 3 (NLRP3) after H/R. However, activation of NF-κB by exogenous administration of phorbol 12-myristate 13-acetate (PMA), even with simultaneous TXNIP knockdown, re-activated the inflammatory pathway and cancelled the aforementioned cardio-protective effects.

In the second study, the application of rNLIP for three days induced cardio-protection in both non-diabetic and diabetic rats with significantly smaller infarct size and less apoptosis after myocardial IRI. Although diabetic rats had a dramatic increase in TXNIP levels after myocardial IRI, rNLIP attenuated the up-regulation of TXNIP and NLRP3. The phosphorylation of IκBα/NF-κB and expression of cytokines after myocardial IRI were also suppressed by rNLIP, even in diabetic rats.

In the third study, Rap1 knockout in the mice effectively repressed the activation of IκBα/NF-κB signaling-mediated inflammation after myocardial IRI. However, Rap1 knockdown in H9C2 cells failed to inhibit this inflammatory pathway. Rap1 deficiency inhibited NLRP3 activation in re-perfused ischemic heart and in LPS and ATP-treated THP-1 macrophages. Moreover, knockdown of Rap1 within LPS-stimulated THP-1 macrophages greatly reduced LDH levels in co-cultured H9C2 cells after H/R.

To conclude, by up-regulating inflammation, TXNIP may be responsible for the aggravation of myocardial IRI in hyperglycemic conditions and, hence, TXNIP inhibition can be cardio-protective. In addition, rNLIP could protect the diabetic heart from myocardial IRI, possibly via suppressing TXNIP and relevant inflammatory pathways. Lastly, this thesis found Rap1 deficiency could inhibit IκBα/NF-κB signaling during myocardial IRI and suppress inflammation in macrophages. These findings might enhance the understanding of myocardial IRI occurring via inflammation-related mechanisms and hopefully assist in developing practicable treatment.