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. 2016 Feb 16:4:e1706.
doi: 10.7717/peerj.1706. eCollection 2016.

Ischemic postconditioning influences electron transport chain protein turnover in Langendorff-perfused rat hearts

Song Cao  1 , Yun Liu  2 , Haiying Wang  3 , Xiaowen Mao  4 , Jincong Chen  3 , Jiming Liu  3 , Zhengyuan Xia  4 , Lin Zhang  5 , Xingkui Liu  3 , Tian Yu  5
Affiliations

Ischemic postconditioning influences electron transport chain protein turnover in Langendorff-perfused rat hearts

Song Cao et al. PeerJ. .

Abstract

Ischemia postconditioning (IPo) is a promising strategy in reducing myocardial ischemia reperfusion (I/R) injury (MIRI), but its specific molecular mechanism is incompletely understood. Langendorff-perfused isolated rat hearts were subjected to global I/R and received IPo in the absence or presence of the mitochondrial ATP-sensitive potassium channel (mitoKATP) blocker 5-hydroxydecanoate (5-HD). Myocardial mitochondria were extracted and mitochondrial comparative proteomics was analyzed. IPo significantly reduces post-ischemic myocardial infarction and improved cardiac function in I/R rat hearts, while 5-HD basically cancelled IPo's myocardial protective effect. Joint application of two-dimensional polyacrylamide gel electrophoresis (2DE) and MALDI-TOF MS identified eight differentially expressed proteins between groups. Expression of cardiac succinate dehydrogenase (ubiquinone) flavoprotein subunit (SDHA) increased more than two-fold after I/R, while IPo led to overexpression of dihydrolipoyl dehydrogenase (DLD), NADH dehydrogenase (ubiquinone) flavoprotein 1 and isoform CRA_b (NDUFV1). When the mitoKATP was blocked, MICOS complex subunit Mic60 (IMMT) and Stress-70 protein (Grp75) were over expressed, while DLDH, ATPase subunit A (ATPA) and rCG44606 were decreased. Seven of the differential proteins belong to electron transport chain (ETC) or metabolism regulating proteins, and five of them were induced by closing mitoKATP in I/R hearts. We thus conclude that IPo's myocardial protective effect relies on energy homeostasis regulation. DLD, SDHA, NDUFV1, Grp75, ATPA and rCG44606 may contribute to IPo's cardial protective effect.

Keywords: Comparative proteomics; Electron transport chain; Energy homeostasis; Ischemia-reperfusion (I/R) injury; Mitochondrial ATP-sensitive potassium channel (mitoKATP); Postconditioning.

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Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. Reperfusion protocol.
Rats were allocated into 4 groups: normal (Nor), I/R (I/R), IPo (IPo) and 5-hydroxydecanoate + IPo (5-HD). Rat hearts were equilibrated for at least 20 min before the application of above protocols. Excluding the Nor group, all hearts underwent ischemia for 40 min (indicated with black area). The I/R group were exposed to 40 min ischemia alone. The hearts in IPo group underwent IPo (palisade white and black bars) alone while 5-HD was administrated in the first 3 min of reperfusion in 5-HD group.
Figure 2
Figure 2. Comparison of hemodynamic parameters and infarct size.
IPo effectively reduced myocardial dysfunction (A–D) and infarct size (E) induced by MIRI while 5-HD partially conteracted IPo’s effects on hemodynamic parameters and infarct size. n = 6 (A–D) or 4 (E) in each group. ∗P < 0.05.
Figure 3
Figure 3. Transmission electron microscopy analysis of myocardium damage.
TEM of Langendorff-perfused myocardium at the end of reperfusion in Control group (A), I/R group (B), IPo group (C) and 5-HD group (D). (E) Flameng’s score of mitochondria from electron microscopy. n = 100 for each group, ∗P < 0.05.
Figure 4
Figure 4. Flow chart of mitochondria preparation.
Pure mitochondria are collected with Nycodenz density gradient centrifugation (A) and representative transmission electron micrograph of the isolated purified mitochondria (C). (B) Crude mitochondria. (C) Sample came from 23% to 25% layer of Nycodenz. (D) Sample isolated from the 25% to 30% layer Nycodenz. Scale bars represent 2 µm.
Figure 5
Figure 5. Proteomic comparison of differential proteins from rat mitochondria.
Langendorff hearts were treated with 5-HD (A) or IPo (B) after I/R insult. Total mitochondrial protein samples were separated using linear IPG strips (24 cm, pH 5–8), and stained with silver nitrate. (C) Protein spots with differential parts per million (PPM) value were annotated with numbers in an image synthesized by PDQuest software. Protein in spot 3002 (detected as ATPA by MASCOT peptide mass fingerprint (PMF) and indicated with a black arrow) was detected as a differential protein by PDQuest. (D) Enlarged 2DE gel images and 3D view showing the 5-HD- and IPo-dependent protein expression changes of ATPA.
Figure 6
Figure 6. Expression trends of DLD, GRP5 and NDUFV1.
Compared with the I/R group, NDUFV1 and DLD increased relatively in IPo group (A, B, E); Compared with the IPo group, GRP75 up-regulated while DLD down-regulated in the 5-HD group (C, D, F). Three replicates for each group. ∗P < 0.05.
See this image and copyright information in PMC

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