Central role of mitofusin 2 in autophagosome-lysosome fusion in cardiomyocytes

Abstract

In the heart, autophagy has been implicated in cardioprotection and ischemia-reperfusion tolerance, and the dysregulation of autophagy is associated with the development of heart failure. Mitochondrial dynamic proteins are profoundly involved in autophagic processes, especially the initiation and formation of autophagosomes, but it is not clear whether they play any role in cardiac autophagy. We previously reported that mitofusin 2 (MFN2), a mitochondrial outer membrane protein, serves as a major determinant of cardiomyocyte apoptosis mediated by oxidative stress. Here, we reveal a novel and essential role of MFN2 in mediating cardiac autophagy. We found that specific deletion of MFN2 in cardiomyocytes caused extensive accumulation of autophagosomes. In particular, the fusion of autophagosomes with lysosomes, a critical step in autophagic degradation, was markedly retarded without altering the formation of autophagosomes and lysosomes in response to ischemia-reperfusion stress. Importantly, MFN2 co-immunoprecipitated with RAB7 in the heart, and starvation further increased it. Knockdown of MFN2 by shRNA prevented, whereas re-expression of MFN2 restored, the autophagosome-lysosome fusion in neonatal cardiomyocytes. Hearts from cardiac-specific MFN2 knock-out mice had abnormal mitochondrial and cellular metabolism and were vulnerable to ischemia-reperfusion challenge. Our study defined a novel and essential role of MFN2 in the cardiac autophagic process by mediating the maturation of autophagy at the phase of autophagosome-lysosome fusion; deficiency of MFN2 caused multiple molecular and functional defects that undermined cardiac reserve and gradually led to cardiac vulnerability and dysfunction.

FIGURE 1.

MFN2 depletion causes autophagosome accumulation. A, representative multimembrane structures (arrows) and autophagic vacuoles (arrowheads) in CKO mouse heart at 4 months of age by TEM. Scale bar, 1 μm. B, increased LC3-II level. n = 3 pairs. C, immunohistochemical staining of endogenous LC3 in wild-type and CKO heart. Scale bar, 50 μm. D, immunofluorescence staining of endogenous LC3 in isolated wild-type and CKO cardiomyocytes. Scale bar, 20 μm. *, significant difference between wild-type and MFN2 CKO hearts.

FIGURE 2.

Defective autophagic degradation in MFN2 CKO heart. A, increased p62 level by Western blot and statistics of the p62/tubulin ratio. n = 3 pairs of wild-type control and MFN2 CKO mice at 4 months old. B, LC3-II protein levels showing the autophagic flux determined by Western blot in Langendorff-perfused hearts under ischemia-reperfusion stress (IR) with or without the lysosomal inhibitors NH₄Cl and pepstatin A. C, LC3-II/tubulin ratios from B. n = 3 for each group. D, confocal images of Langendorff-perfused hearts stained by LysoTracker^TM red before and after ischemia-reperfusion stress. E, surface area of LysoTracker^TM red staining counted from D. *, significant difference between wild-type and MFN2 CKO hearts; †, significant difference between inhibitor-treated and untreated groups; #, significant difference between ischemia-reperfusion (I/R)-treated and untreated groups.

FIGURE 3.

MFN2 depletion increases ER stress signals. A, confocal images of adult cardiomyocytes transfected with Ad-MFN2-GFP for 48 h and then loaded with MitoTracker. Scale bars are as follows: 10 μm for the whole cell and 1 μm for the high magnification view. B, cardiomyocytes from control (upper) and MFN2 CKO (middle) mouse heart stained with ER-Tracker^TM and MitoTracker^TM. High magnification views (bottom) of ER-Tracker^TM staining in MFN2 CKO cardiomyocytes. Scale bars are as follows: 20 μm for top and middle and 5 μm for bottom. C, mRNA levels of ER stress-related genes. D, expression of phospho-eIF2α and total-eIF2α in wild-type and CKO hearts. E, confocal images of neonatal cardiomyocytes transfected with Ad-GFP-LC3, treated with 1 μm thapsigargin or 5 μg/ml tunicamycin for 5 h, and loaded with LysoTracker^TM for 10 min before imaging. F, relative fold change of merged LC3 dots and LysoTracker^TM dots in cells treated with thapsigargin (1 μm), tunicamycin (5 μg/ml), or bafilomycin A1 (50 nm) compared with cells treated with DMSO. n = 35 cells for each group from three independent experiments. *, significant difference between control and treated cells.

FIGURE 4.

Regulation of autophagosome-lysosome fusion by MFN2. A, Western blotting showing MFN2 protein levels in neonatal cardiomyocytes transfected with adenovirus containing scrambled RNA, MFN2 shRNA1, MFN2 shRAN2, or MFN2 shRNA2 co-transfected adenovirus containing MFN2 cDNA. B, confocal images of neonatal cardiomyocytes co-transfected with Ad-GFP-LC3 and indicated adenovirus for 60 h and then loaded with LysoTracker^TM red. Scale bar, 10 μm. C, percentages of LC3 dots merged with lysosomal dots in total LC3 dots in neonatal cardiomyocytes in B or D in the presence of rapamycin. n = 35 cells from three independent experiments. *, significant difference between scrambled and MFN2 knockdown neonatal cardiomyocytes. E, expression levels of FYCO1, RAB7, and MFN2 in wild-type and CKO hearts. Tubulin was used as loading control. F, whole heart lysates from mice with or without starvation were immunoprecipitated with anti-RAB7 or anti-MFN2 and analyzed with MFN2 or RAB7 antibodies, respectively. IgG was used as negative immunoprecipitation control. IP, immunoprecipitation; IB, immunoblot.

FIGURE 5.

Cell metabolism and cardiac function of MFN2 CKO mice. A, BODIPY 493/503 staining for lipid droplets in isolated wild-type and CKO cardiomyocytes from 4-month-old mice. Insets show magnified droplets. Scale bar, 20 μm; inset scale bar, 2 μm. B, periodic acid-Schiff staining (scale bar, 50 μm); C, TEM (scale bar, 2 μm) for lipofuscin in wild-type and CKO hearts. D, mitochondrial respiratory control ratios in mitochondria isolated from wild-type and CKO hearts. E, state III O₂ consumption and state IV O₂ consumption. n = 6 pairs of mice at 4 months of age. F, echocardiogram measurements of cardiac function reflected by ejection fraction from MFN2 CKO mice and control littermates at 4, 6, 12 and 17 months of age. n = 18 pairs. G, representative confocal images of mitochondrial Δψ_m stained by tetramethylrhodamine methyl ester in Langendorff-perfused hearts before or after I/R stress from 6-month-old MFN2 CKO, Mfn2 littermate control (WT), and Mfn2^+/+ Mlc2v-Cre (Cre) mice. Scale bar, 50 μm. H, statistics of percentages of cardiomyocytes maintaining Δψ_m before or after I/R stress in perfused mouse hearts, at 6 months of age. n = 4 hearts with 200 image frames for each group. *, significant difference between MFN2 CKO and littermate control or Mlc2v-Cre control.