Proteomic and metabolomic characterization of cardiac tissue in acute myocardial ischemia injury rats.

The pathological process and mechanism of myocardial ischemia (MI) is very complicated, and remains unclear. An integrated proteomic-metabolomics analysis was applied to comprehensively understand the pathological changes and mechanism of MI. Male Sprague-Dawley rats were randomly divided into a mock surgery (MS) group and an MI group. The MI model was made by ligating the left anterior descending coronary artery, twenty-four hours after which, echocardiography was employed to assess left ventricular (LV) function variables. Blood samples and left ventricular tissues were collected for ELISA, metabolomics and proteomics analysis. The results showed that LV function, including ejection fraction (EF) and fractional shortening (FS), was significantly reduced and the level of cTnT in the serum increased after MI. iTRAQ proteomics showed that a total of 169 proteins were altered including 52 and 117 proteins with increased and decreased expression, respectively, which were mainly involved in the following activities: complement and coagulation cascades, tight junction, regulation of actin cytoskeleton, MAPK signaling pathway, endocytosis, NOD-like receptor signaling pathway, as well as phagosome coupled with vitamin digestion and absorption. Altered metabolomic profiling of this transition was mostly enriched in pathways including ABC transporters, glycerophospholipid metabolism, protein digestion and absorption and aminoacyl-tRNA biosynthesis. The integrated metabolomics and proteomics analysis indicated that myocardial injury after MI is closely related to several metabolic pathways, especially energy metabolism, amino acid metabolism, vascular smooth muscle contraction, gap junction and neuroactive ligand-receptor interaction. These findings may contribute to understanding the mechanism of MI and have implication for new therapeutic targets.

Effect of moxibustion preconditioning on autophagy-related proteins in rats with myocardial ischemia reperfusion injury.

BACKGROUND: Autophagy has increasingly been recognized as playing an essential role in the pathogenesis of myocardial ischemia reperfusion injury (MIRI). Moxibustion, a form of heat therapy commonly used in traditional Chinese medicine (TCM), has been shown to exhibit cardioprotective effects. However, whether the cardioprotective effect of moxibustion is related to the regulation of autophagy remains unknown. This study aimed to investigate the possible mechanism underlying the cardioprotective effect of moxibustion preconditioning at PC6 on MIRI by measuring the expressions of proteins involved in the regulation of autophagy. METHODS: Male Sprague-Dawley rats were randomly divided to receive moxibustion preconditioning or autophagy inhibitor 3-Methyladenine (3-MA) intervention. Then the MIRI model was established by ligating the left anterior descending (LAD) coronary artery for 30 minutes followed by reperfusion for 4 hours. After 4 hours of reperfusion, the myocardial infarction area was assessed using Evans blue and TTC staining, and cTnT and lactate dehydrogenase (LDH) levels in the serum were determined by ELISA. Hematoxylin and eosin (H&E) staining was performed for morphological evaluation of ventricular tissues. Expressions of autophagy components Beclin 1, Bcl-2, and Akt were assessed using quantitative real-time PCR, immunohistochemistry (IHC) and western blot. RESULTS: Moxibustion preconditioning significantly reduced the necrotic area and the levels of cTnT and LDH were similar to the 3-MA intervention, also attenuated morphological alterations were induced by MIRI. Simultaneously, the mRNA and protein expressions of Beclin 1 and Akt were up-regulated, while those of Bcl-2 were down-regulated by MIRI. Moxibustion preconditioning and 3-MA intervention reversed MIRI-induced changes in Beclin 1, Akt, and Bcl-2 expressions. CONCLUSIONS: Moxibustion preconditioning at PC6 can attenuate myocardial injury for MIRI in a similar way to 3-MA intervention. This cardioprotective effect of moxibustion preconditioning may be mediated by modulating autophagy via regulation of Beclin 1, Bcl-2 and Akt.

Changes in the Serum Metabolome of Acute Myocardial Ischemia Rat Pretreatment with Electroacupuncture.

Myocardial infarction (MI), the most common symptom is chest pain, occurs when blood flow decreases or stops to a part of the heart, causing damage to the heart muscle. Electroacupuncture pretreatment (EP) is a recent observation which has been shown to induce ischemic tolerance like the ischemia preconditioning, suggesting that EP may be a promising preventive strategy for individual susceptibility to MI. This study investigated mechanisms that underlie the effect of EP on MI through the use of gas chromatography-mass spectrometry (GC-MS)-based metabolic profiling. Male Sprague-Dawley rats were randomly divided to receive or not receive three days of EP at PC6 (Neiguan). Then on the fourth day, each group was further divided to undergo mock surgery or MI, induced by ligation of the left anterior descending coronary artery. After 24h, the blood samples and hearts were collected for the follow-up research. The results showed that treatment by EP significantly reduced the levels of CK-MB, cTnT, AST, and MDH in serum and decreased myocardial infarction area. According to GC-MS-based serum metabolic profiling and analysis, a total of 636 characteristic peaks were identified, including 158 known and 478 unknown metabolites. MI caused comprehensive metabolic changes in glycolysis-related metabolites, malate-aspartate shuttle (MAS) metabolites, and purine metabolites with anti-oxidant functions, while EP reversed more than half of the differential metabolic changes, mainly affecting amino acid and energy metabolism, especially the glutamate metabolism and MAS. In a word, our findings suggest that EP exerts its cardioprotective effect on MI by regulating amino acid and energy metabolisms. Meanwhile, GC-MS-based metabolomics provided a powerful way to characterize the metabolic features of MI, with and without EP, and thereby improved our understanding of the effect and mechanisms of EP.

[Time-dependent Protective Effect of Electroacupuncture on Ischemic Myocardium and Changes of Myocardial Autophagy and Apoptosis Related Protein Expression in Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at different time-points on the injured myocar-dium and expression of myocardial Bax/Bcl-2 and Lc 3 Ⅱ/Ⅰ proteins in acute myocardial ischemia-reperfusion injury (MIRI) rats so as to explore its mechanisms underlying myocardial protective effect via reducing cardiomyocyte autophagy and apoptosis. METHODS: A total of 66 adult SD rats were randomly divided into sham operation (sham), model, EA-R 0min(R＝ reperfusion), EA-R 30min, EA-R 60min, and EA-R 120min groups, with 6 rats being in the sham group and 12 rats being in each of the other 5 groups. The MIRI model was prepared by ligating the anterior descending branch (ADB) of the left coronary artery for 30 min followed by reperfusion for 4 h. In the sham group, the ADB was only threaded without ligation. EA was applied to bilateral "Neiguan" (PC 6) for 20 min at 0, 30, 60, and 120 min when reperfusion. Evans Blue-triphenyltetrazolium chloride (TTC) double staining was performed to determine the myocardial infarction area (MIA) and the ratio of the infarct size of the area at risk (IS/AAR). ELISA was performed to measure serum cardiac troponin 1 (cTn-Ⅰ) content, and Western blot was used to measure the expression of apoptosis-related proteins Bax and Bcl-2 and autophagy related proteins Lc 3 Ⅱ and Lc 3 Ⅰ in the left cardiac ventricle tissue. RESULTS: Compared with the model group, the percentages of MIA in the EA-R 30min, EA-R 60min, and EA-R 120min groups, and the IS/AAR in the EA-R 0min, EA-R 30min, EA-R 60min and EA-R 120min groups were significantly reduced (P<0.05, P<0.01). Comparison among the 4 EA groups showed that the percentages of MIA and the IS/AAR were considerably lower in the EA-R 30min, EA-R 60min, and EA-R 120min groups than in the EA-R 0min group (P<0.05, except IS/AAR in the EA-R 120min group), but significantly higher in the EA-R 60min and EA-R 120min groups than in the EA-R 30min group (P<0.05, P<0.01), suggesting a better therapeutic effect of EA intervention at 30 min of MIRI in improving MI. In comparison with the sham group, myocardial cTn-Ⅰ content and Bax/Bcl-2 and Lc 3 Ⅱ/Ⅰ levels in the model group were significantly increased (P<0.01). After EA intervention, the increased cTn-Ⅰ content and Bax/Bcl-2 and Lc 3 Ⅱ/Ⅰ levels in the EA-R 0min, EA-R 30min, EA-R 60min, and EA-R 120min groups were significantly reduced (P<0.05, P<0.01). The cTn-Ⅰ content was obviously lower at EA-R 30 min, but markedly increased at EA-R 120min than at EA-R 0min (P<0.05). The expression of Bcl-2 was obviously higher at EA-R 30min than at EA-R 0min (P<0.05). No significant differences were found among the 4 EA intervention time-points in the levels of Bax/Bcl-2 and Lc 3 Ⅱ/Ⅰ (P>0.05). CONCLUSION: EA intervention can reduce MIA in MIRI rats, which is possibly closely related to its effects in reducing apoptosis and autophagy. The best intervention time is at 30 min after MI reperfusion, but the difference of effects of EA at different time-points is independent of Bax/Bcl-2 and Lc 3 Ⅱ/Ⅰ expression.

[Effect of Moxibustion Preconditioning with Seed-sized Moxa Cones on Myocardial Infarction Size and Beclin 1 Expression in Myocardial Ischemia-reperfusion Injury Rats].

OBJECTIVE: To observe the effect of moxibustion (Moxi) preconditioning with seed-sized moxa cones on myocardial ischemia-reperfusion injury (MI/RI) at different stages, and to analyze the correlation between this effect and the expression of autophagy related protein Beclin 1. METHODS: This study contains two parts: 1) changes of myocardial pathological injury and percentages of myocardial infarcted area at different time-points after modeling and Moxi intervention, and 2) effect of Moxi on contents of serum cardiac troponin T(cTnT) and expression of myocardial Bcl-2, Bax and Beclin 1 proteins. In the first part, 42 SD rats were randomly divided into model group, 1 day (d) Moxi group, 2 d Moxi group, 3 d Moxi group，4 d Moxi group, 5 d Moxi group and 7 d Moxi group. The model of MI/RI was established by ligating the left anterior descending coronary artery (LAD) for 30 min and reperfusion for 240 min. The electrocardiogram (ECG) of standard limb lead Ⅱ was monitored and the heart was taken 4 h after reperfusion for examining myocardial infarcted size with triphenyl tetrazolium chloride (TTC) staining. In the second part, 48 SD rats were randomized into sham-operation, model, moxibustion and autophagy inhibitor (3-MA) groups, with 12 rats in each group. The serum cTnT level was assayed and histopathological changes of the myocardial tissue below the ligation site were examined with HE staining, and the expression levels of Bcl-2, Bax and Beclin 1 proteins in the myocardial tissue below the LAD-ligated site were detected using Western blot. RESULTS: Compared with the model group, the percentages of myocardial infarcted area were significantly decreased in the 4 d, 5 d and 7 d Moxi groups (P<0.05), but without significant differences among the 3 Moxi groups (P>0.05). The state of MI-induced breakage and disordered arrangement of myocardial fibers with interstitial edema and inflammatory cell infiltration at the MI stage in the Moxi group and at the reperfusion stage in the autophagy inhibitor group was relatively lighter. The levels of serum cTnT content and Bax/Bcl-2 and Beclin 1 protein expression at the MI and reperfusion stages were significantly higher in the model group than in the sham-operation group (P<0.01), and considerably lower in the Moxi and autophagy groups than in the model group (P<0.01). The serum cTnT content, ratio of Bax/Bcl-2 expression and Beclin 1 expression levels at the MI and reperfusion stages were significantly lower in the autophagy inhibitor group than in the Moxi group (P<0.05). CONCLUSIONS: Moxibustion with seed-sized moxa cones at "Neiguan" (PC 6) can effectively alleviate myocardial ischemia in MI/RI rats, which is probably related to its effect in down-regulating Bax/Bcl-2 and Beclin 1 expression and in inhibiting autophagy.