TXNIP Increase Autophagy Through AMPK During Myocardial Ischemia/reperfusion Injury

BackgroundRestoring blood flow during ischemia plays a crucial role in limiting myocardial infarct size and maintaining cardiac function. However, it can also trigger adverse effects that damage myocardium and amplifycontractile dysfunction known as ischemia reperfusion injury. Researches have shown that the excessively activated autophagy during reperfusion contributed to reperfusion injury.Autophagy is an intracellular bulk degradation process for proteins and organelles. In the heart, autophagy is stimulated by both myocardial ischemia and ischemia reperfusion. Autophagy plays bilateral roles during ischemia and reperfusion. Autophagy is protective during ischemia, whereas it is detrimental during reperfusion. However, the mechanismof whythe autophagy is excessively activated during reperfusion was still unclear.TXNIP, known as thioredoxin interacting protein, is increased during MI/R. Previous reports shown that suggest that TXNIP is involved directly in a key switch from aerobic to anaerobic metabolism by regulating mitochondrial respiration and PDH activity. Thus, TXNIP-KO hearts hadgreater recovery of cardiac function after an ischemia-reperfusion insult. In response to changes in the intracellular ATP/AMP ratio, AMPK is activated and leads to autophagy. Since TXNIP is increased during MI/R and can also regulated cardiomyocyte energy hemostasis, whether TXNIP is capable in regulating autophagy was still unclear. Therefore, in the present study, we aim to elucidate a possible relationship between increased TXNIP and excessively activated autophagy during MI/R injury.Aims1. To determine whether knockout or overexpressing TXNIP affects myocardium injury and cardiac dysfunction during MI/R.2. To investigate whether knockout or overexpressing TXNIPchanges autophagy.3. To elucidate the possible pathway that involved in the regulation of autophagy by TXNIP during MI/R.Methods1. Observing whether knockout or overexpressing TXNIP aggravated MI/R injury1.1 Breeding and genotyping TXNIPfloxed/floxedMyosin6-Cre mice1.2 Establishing TXNIP overexpressed mice by cardiac specific injecting adenovirus1.3 Performing MI/R surgeryMice were anesthetized and MI was induced after temporarily exteriorizing the heart via a left thoracic incision at the fourth intercostal space and subsequently tying a 6-0 silk slipknot around the left anterior descending coronary artery. After 40 minutes of ischemia, the slipknot was released. Mice were sacrificed at indicated time points. Samples for Western blot were collected at the end of 40 min MI, 1.5 and 3hr after MI/R. Apoptosis was elevated3 hr after MI/R, and cardiac function and infarct size assessment were determined at 24 hr after MI/R. Sham group went under the same procedure but the slipknot was not tied1.4 Determine TXNIP expression by Western blot1.5Cardiac function was determined by echocardiography and invasive hemodynamic evaluation methods at the end of 24 hours reperfusion period1.6 Myocardial infarct size was determined by the Evans blue/TTC double staining1.7 Cardiac apoptosis was observed by TUNEL staining2 Observingwhether knockout or overexpressing TXNIP changes autophagy level2.1 Genotyping LC3-GFP transgenic mice, breed TXNIP Myosin6-Cre-LC3-GFP mice, and establishing LC3-GFP-TXNIP overexpressed mice by cardiac specific injecting adenovirus2.2 Performing MI/R surgeryThe procedure was same above. Samples for Western blot were collected at the end of 40 min MI and 3hr after MI/R. Autophagy was elevated3 hr after MI/R by transmission electron microscopy and fluorescence microscopy2.3 Determine LC3 and P62 expression by Western blot2.4 Imaging autophagosome in myocardium by transmission electron microscopy2.5 Observing LC3-GFP dots by fluorescence microscopy3 TXNIP increases autophagy through AMPK3.1 Genotyping AMPKfloxed/floxedMyosin6-Cre mice, and establishing AMPKfloxed/floxedMyosin6-Cre-TXNIP overexpressed mice3.2 Performing MI/R surgery. The procedure was same above. Samples for ATP assay and Western blot were collected at the end of 40 min MI and 3hr after MI/R. Autophagy was elevated3 hr after MI/R by transmission electron microscopy3.3 Determine ATP content in myocardium3.4 Determine pAMPK, AMPK, pRaptor, Raptor, pULK1 and ULK1 expression by Western blot3.5 Imaging autophagosome in myocardium by transmission electron microscopy3.6 Determine LC3 expression by Western blotResults1. Elevated TXNIP expression by MI/R aggravated MI/R injury1.1 TXNIP expression is significantly increased during MI and MI/R1.2 TXNIP overexpression aggravated MI/R-induced cardiac dysfunctionTXNIP overexpressed mice showed significantly decreased LVEF% and worser LVEDP,+dp/dtmax and-dp/dtmax compare with WT mice. However, TXNIP KO mice showed cardiac protective effects.1.3 TXNIP overexpression increased infarct size and apoptosisCompare with WT, TXNIP overexpressed mice showed significantly larger infarct size and apoptosis2 Elevated TXNIP enhanced MI/R-induced autophagy activation2.1 Elevated TXNIP affected LC3 II/I ratioDuring MI/R, the ratio of LC3 II/I in TXNIP overexpressed mice was significantly higher than in WT and TXNIP KO mice. However, LC3 II/I ratio in TXNIP KO mice was significantly lower than in WT mice2.2 Elevated TXNIP increased MI/R-induced autophagosome numberBy transmission electron microscopy and fluorescence microscopy, we found the autophagosomenumber in TXNIP KO mice was significantly lesser than in WT, the autophagosomenumber in TXNIP overexpressed mice was larger the in WT3 TXNIP increase autophagy through AMPK3.1 TXNIP expressions affected ATP productionCompare with WT, ATP content was higher in TXNIP KO mice. The ATP content was decreased in TXNIP overexpressed mice compare with WT mice3.2 TXNIP regulated autophagy through AMPKIn TXNIP overexpressed mice, the ratio of pAMPK/AMPK and pRaptor/Raptor was increased, but the ratio was dropped in TXNIP KO mice. In AMPK KO mice, compare with WT mice, overexpressing TXNIP had lesser effects in increasing autophagosomenumber and LC3 II/I ratioConclusion1. Elevated TXNIP during MI/R aggravated MI/R-induced cardiac dysfunction, enlarge infarct size and increase apoptosis. Knockout TXNIP have the opposite effects.2. TXNIP increased autophagy during reperfusion through AMPK. These results provided us more insight of the regulation of autophagy during MI/R.