Melatonin protects cardiac microvasculature against ischemia/reperfusion injury via suppression of mitochondrial fission-VDAC1-HK2-mPTP-mitophagy axis

Abstract

The cardiac microvascular system, which is primarily composed of monolayer endothelial cells, is the site of blood supply and nutrient exchange to cardiomyocytes. However, microvascular ischemia/reperfusion injury (IRI) following percutaneous coronary intervention is a woefully neglected topic, and few strategies are available to reverse such pathologies. Here, we studied the effects of melatonin on microcirculation IRI and elucidated the underlying mechanism. Melatonin markedly reduced infarcted area, improved cardiac function, restored blood flow, and lower microcirculation perfusion defects. Histological analysis showed that cardiac microcirculation endothelial cells (CMEC) in melatonin-treated mice had an unbroken endothelial barrier, increased endothelial nitric oxide synthase expression, unobstructed lumen, reduced inflammatory cell infiltration, and less endothelial damage. In contrast, AMP-activated protein kinase α (AMPKα) deficiency abolished the beneficial effects of melatonin on microvasculature. In vitro, IRI activated dynamin-related protein 1 (Drp1)-dependent mitochondrial fission, which subsequently induced voltage-dependent anion channel 1 (VDAC1) oligomerization, hexokinase 2 (HK2) liberation, mitochondrial permeability transition pore (mPTP) opening, PINK1/Parkin upregulation, and ultimately mitophagy-mediated CMEC death. However, melatonin strengthened CMEC survival via activation of AMPKα, followed by p-Drp1S616 downregulation and p-Drp1S37 upregulation, which blunted Drp1-dependent mitochondrial fission. Suppression of mitochondrial fission by melatonin recovered VDAC1-HK2 interaction that prevented mPTP opening and PINK1/Parkin activation, eventually blocking mitophagy-mediated cellular death. In summary, this study confirmed that melatonin protects cardiac microvasculature against IRI. The underlying mechanism may be attributed to the inhibitory effects of melatonin on mitochondrial fission-VDAC1-HK2-mPTP-mitophagy axis via activation of AMPKα.

Keywords: AMP-activated protein kinase α; cardiac microcirculation endothelial cells; hexokinase 22; melatonin; microvascular IR injury; mitochondrial fission; mitochondrial permeability transition pore opening; voltage-dependent anion channel 1.

© 2017 The Authors. Journal of Pineal Research Published by John Wiley & Sons Ltd.

Figures

Figure 1

Melatonin reduced infarct size and preserved cardiac function following ischemia/reperfusion injury in vivo which was performed by 30 min of ischemia followed by 2‐h reperfusion (n=6/group). (A). Representative pictures of heart sections with TTC and Evans Blue staining. (B). Bar graph indicates the infarct size expressed as a percentage of the total left ventricular area. The lactate dehydrogenase release, troponin T contents, and CK‐MB values were assessed via ELISA. (C). Representative M‐mode echocardiograms was performed after 2‐h reperfusion with the parasternal long‐axis views in each group. (D). Quantitative analysis of cardiac function by echocardiography. *P<.05 vs sham group; #P<.05 vs Ctrl‐IRI group, &P<.05 vs Mel‐IRI group

Figure 2

Melatonin protected acute microcirculation malfunction during ischemia/reperfusion injury(IRI) via AMPKα. (A‐B). Representative case of a myocardial contrast echocardiography myocardial contrast echocardiography parasternal long‐axis image, which was divided into three regions: apical septum (blue), mid‐septum (yellow), and basal septum (red). Left ventricular: Left ventricle. A: plateau intensity, β: flow velocity. (C). Myocardial blood flow (A×β) profiles in the apical region for different groups at 15, 45, 75, and 120 min after reperfusion. (D). Apical myocardial blood flow 120 min after reperfusion. n=6 per group. (E). Microvascular image detection by ink staining. (F). Immunohistochemistry of endothelial nitric oxide synthaseS expression. (G). HE staining for red blood cell morphology in different groups. (H). Transmission electron microscopy (TEM) was used to observe the structural changes of microvessel in response to IRI, including microvascular wall destruction and luminal stenosis (white arrow) of CMEC. *P<.05 vs sham group, #P<.05 vs Ctrl‐IRI group, &P<.05 vs Mel‐IRI group

Figure 3

Melatonin maintained microvessels endothelial barrier integrity, reduced vascular permeability, and alleviated cellular death. (A). The endothelial barrier integrity was assessed via VE‐cadherin staining. Discontinuous punctiform or linear expression of VE‐cadherin could be observed in the ischemia/reperfusion injury(IRI) group indicative of the broken endothelial barrier. However, melatonin could reverse the continuous linear of VE‐cadherin fluorescence via AMPKα. (B). The expression of ICAM1 in cardiac microcirculation endothelial cells(CMEC). (C). The Gr‐1+ neutrophil infiltration into the myocardial tissues. (D). The leakage of plasma albumin out of the surface of the vessel wall into interstitial spaces suggested the increased microvascular permeability in response to IRI. (E). TUNEL assay to assess CMEC death

Figure 4

Ischemia/reperfusion injury induced(IRI) cardiac microcirculation endothelial cells(CMEC) death via excessive mitophagy in vitro. Wild‐type mice‐ and AMPKα−/− mice‐derived CMEC treated with melatonin were named Mel+WT and Mel+AMPKα−/− group, respectively. Furthermore, AMPKα gain‐of‐function experiments was performed in CMECs from AMPKα−/− using adenovirus vector following melatonin treatment, named Ad+Mel+AMPKα−/− group. Meanwhile, rapamycin (Rap), an activator of mitophagy, was used in Mel+WT as the positive control group. The IRI in vitro was mimicked by 30 min of hypoxia with serum starvation and 2 h of reoxygeneration. (A). Transmission electron microscope was performed to observe mitophagy in vivo. IRI induced more mitochondria were contained by lysosome, and these changes were reversed by melatonin. Yellow arrow suggested the normal mitochondria. White arrow indicated the digested mitochondria or fragmented mitochondria in lysosome. (B). The co‐location of lysosome and mitochondria. More fragmented mitochondria were consumed by lysosomes in response to IRI, which was reversed by melatonin. Rap reduced more numbers of mitochondria contained in lysosome. (C). The change of proteins related to mitophagy. IRI increased the ratio of LC3 II/LC3 I via PINK1/Parkin pathway activation. (D‐E). TUNEL staining demonstrated that mitophagy evoked CMEC death. (F). The lactate dehydrogenase release assay to detect cellular damage. 3‐MA, an inhibitor of mitophagy. *P<.05 vs Ctrl group, #P<.05 vs IRI‐WT group, &P<.05 vs IRI‐Mel+WT group, @P<.05 vs IRI‐Mel+ AMPKα−/− group

Figure 5

Mitophagy was activated by mitochondrial permeability transition pore(mPTP) opening that was induced by hexokinase 2(HK2) separation from the mitochondria in response to Ischemia/reperfusion injury induced(IRI). (A‐B). The change of membrane potential (∆Ψm) by JC‐1 staining. (C.) Change in mPTP opening. (D‐H). The subcellular location of HK2 via immunofluorescence and Western blots. IRI contributed to the HK2 separation from the mitochondria to the cytoplasm, which was reversed by melatonin via AMPKα−/−. However, IRI or melatonin had no effects on the total content of HK2 but influenced its subcellular distribution between the mitochondria and cytoplasm. (I‐J). Arbitrary mPTP opening time by tetramethylrhodamine ethyl ester(TMRE) fluorescence of 3–Brpa which is the inhibitors of V HK2 interaction. Arbitrary mPTP opening time was determined as the time when the TMRE fluorescence intensity decreased by half between the initial and residual fluorescence intensity. CsA, an mPTP blocker, was used as the negative control. (K). The co‐expression of Parkin and mitochondria. Inhibition of HK2 liberation reduced the interaction of Parkin with mitochondria. *P<.05 vs Ctrl group; #P<.05 vs IRI‐WT group, &P<.05 vs IRI‐Mel+WT group, @P<.05 vs IRI‐Mel+ AMPKα‐/‐ group

Figure 6

Hexokinase 2(Hk2) liberation was attributed to mitochondrial fission‐mediated voltage‐dependent anion channel 1(VDAC1) oligomerization which was inhibited by melatonin via AMPKα. (A‐D). The evaluation of VDAC1 oligomerization via EGS‐based cross‐linking and immunoblotting using anti‐VDAC1 antibodies. Ischemia/reperfusion injury induced increased VDAC1 oligomerization corresponding to molecular masses of 69 and 95 kDa, whereas melatonin can reverse this change. (E). HK2 and VDAC1 interaction assessed by immunoprecipitation (IP) experiments. (F‐H). Inhibition of VDAC1 oligomerization could increase the contents of mitochondria‐HK2. Diphenylamine‐2‐carboxylic acid (DpC), an inhibitor of VDAC1 oligomerization, was used as the negative control group. (I‐J). Mitochondria of cardiac microcirculation endothelial cells were labeled with anti‐Tom20 antibody to determine the number of cells with mitochondria fragmentation. To assess changes in mitochondrial morphology quantitatively, the aspect ratio (AR, the mitochondrial length) and form factor (FF, the degree of mitochondrial branching) were calculated for each cell (the minimum value for both parameters is 1). High FF and AR values shown healthy mitochondria whereas low FF and AR indicated fragmented mitochondria. (K). Inhibition of mitochondrial fission could reverse the VDAC1 expression. Mdivi1, an inhibitor of mitochondrial fission, was used as the negative control group. *P<.05 vs Ctrl group; #P<.05 vs IRI‐WT group, &P<.05 vs IRI‐Mel+WT group, @P<.05 vs IRI‐Mel+ AMPKα−/− group. VDAC1, voltage‐dependent anion channel1; HK2, hexokinase2

Figure 7

Ampkα was activated by melatonin contributed to Drp1 modifications and subsequent mitochondrial fission. (A). Co‐localization of Drp1 and mitochondria. Boxed area under each micrograph represents the amplification of the white square. More Drp1 was located on fragmented mitochondria while melatonin could reduce Drp1 migration on mitochondria via AMPKα. White arrow indicated the aggregated Drp1 on mitochondria or not. (B‐D). Activated AMPKα was involved in regulating Drp1 phosphorylation. *P<.05 vs Ctrl group; #P<.05 vs IRI‐WT group, &P<.05 vs IRI‐Mel+WT group, @P<.05 vs IRI‐Mel+ AMPKα−/− group

Figure 8

(A) Ischemia/reperfusion injury induced(IRI) activated Drp1‐dependent mitochondrial fission, which subsequently induced voltage‐dependent anion channel(VDAC1) oligomerization, hexokinase 2 liberation, mitochondrial permeability transition pore(mPTP) opening, PINK1/Parkin upregulation, and ultimate mitophagy‐mediated cardiac microcirculation endothelial cells(CMEC) death. However, melatonin strengthened CMEC survival via activation of AMPKα, followed by p‐Drp1S637 upregulation and p‐Drp1S16 downregulation, which blunted Drp1‐dependent mitochondrial fission. Suppression of mitochondrial fission by melatonin recovered VDAC1‐HK2 interaction that prevented mPTP opening and PINK1/Parkin activation, eventually blocking mitophagy‐mediated cellular death. (B) Finally, the survival of CMEC preserved barrier integrity, reduced vascular permeability, alleviated neutrophil infiltration and plasma albumin leakage, increased endothelial nitric oxide synthase contents, improved microvessel patency and contributed to microvascular perfusion, protecting cardiac microcirculatory against IRI