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. 2011 Mar;31(6):1309-28.
doi: 10.1128/MCB.00911-10. Epub 2011 Jan 18.

Mitofusin-2 Maintains Mitochondrial Structure and Contributes to Stress-Induced Permeability Transition in Cardiac Myocytes

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Mitofusin-2 Maintains Mitochondrial Structure and Contributes to Stress-Induced Permeability Transition in Cardiac Myocytes

Kyriakos N Papanicolaou et al. Mol Cell Biol. .
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Abstract

Mitofusin-2 (Mfn-2) is a dynamin-like protein that is involved in the rearrangement of the outer mitochondrial membrane. Research using various experimental systems has shown that Mfn-2 is a mediator of mitochondrial fusion, an evolutionarily conserved process responsible for the surveillance of mitochondrial homeostasis. Here, we find that cardiac myocyte mitochondria lacking Mfn-2 are pleiomorphic and have the propensity to become enlarged. Consistent with an underlying mild mitochondrial dysfunction, Mfn-2-deficient mice display modest cardiac hypertrophy accompanied by slight functional deterioration. The absence of Mfn-2 is associated with a marked delay in mitochondrial permeability transition downstream of Ca(2+) stimulation or due to local generation of reactive oxygen species (ROS). Consequently, Mfn-2-deficient adult cardiomyocytes are protected from a number of cell death-inducing stimuli and Mfn-2 knockout hearts display better recovery following reperfusion injury. We conclude that in cardiac myocytes, Mfn-2 controls mitochondrial morphogenesis and serves to predispose cells to mitochondrial permeability transition and to trigger cell death.

Figures

FIG. 1.
FIG. 1.
Cardiac myocyte-specific deletion of Mfn-2. (A) Hearts from mice with the indicated Mfn-2 genotypes (F/F, homozygous for Mfn-2loxP; +/F, heterozygous for Mfn-2loxP) with or without the α-MHC transgene (+ or −) were analyzed by Western blotting. The panel on the right shows the quantification of Mfn-2 band intensity relative to that for tubulin (*, P < 0.05; four samples per group; F/F;− or F/F;cre). (B, left) Gross morphological analysis of F/F;− and F/F;cre hearts to assess cardiac growth and chamber dilation (the scale bars are 1 mm). (Right) Normalization of total heart weight (HW) to body weight (BW) indicates that F/F;cre hearts are moderately but significantly larger than F/F;− or +/+;cre hearts. The numbers of animals are indicated in circles in their respective bars. (C) Microscopic examination of histological sections stained with hematoxylin and eosin to assess myocyte hypertrophy (the scale bars are 50 μm). The lower left panel shows average values in the myocyte cross-sectional area, while the lower right panel shows average values in collagen deposition (*, P was <0.05 for F/F;− versus F/F;cre mice; #, P was <0.05 for +/+;cre versus F/F;cre mice by one-way ANOVA).
FIG. 2.
FIG. 2.
Analysis of cardiac contractility in the presence or absence of Mfn-2. Pressure-volume loop recordings taken before (black line) or after (blue line) acute isoproterenol infusion in hearts with normal levels of Mfn-2 (F/F;−) (A) or in hearts where Mfn-2 had been ablated (F/F;cre) (B). For further details and a quantitative analysis, see also Table 3. (C) Sample [Ca2+]i and shortening records for isolated cardiac myocytes from F/F;− and F/F;cre mice. (D) Representative tracings of the change in [Ca2+]i for F/F;− and F/F;cre myocytes. (E) Average shortening in F/F;− (0.169 ± 0.053) and F/F;cre (0.126 ± 0.035) myocytes (*, P < 0.05, F/F;− versus F/F;cre). (F) Average peak [Ca2+]i in F/F;− (2.85 ± 0.37) and F/F;cre (3.76 ± 0.75) myocytes.
FIG. 3.
FIG. 3.
Electron microscopy analysis of F/F;− and F/F;cre hearts. (A) The typical organization of mitochondria along the myofibrils is detected in F/F;− hearts. (B) Region of an F/F;cre heart containing mostly normal mitochondria. (C) Different region where the mitochondria become abnormally enlarged. (D) Greater detail of a region of an F/F;cre heart containing enlarged spherical mitochondria. (E) In some rare cases, mitochondria are found to display further abnormalities, such as the formation of internal vesicles and crista decondensation. The scale bars are 2 μm. (F) Assessment of mitochondrial (mitoch.) and myofibrillar (myof.) volume density in F/F;− and F/F;cre heart sections using the grid method (44 fields; size, 40 by 13 μm; three hearts per group were analyzed). (G) The area enclosed by the mitochondrial border was quantified in the same samples and averaged for F/F;− and F/F;cre samples. The results indicate a statistically significant increase in mitochondrial area in the F/F;cre group (30 fields from three samples per genotypic group). (H to I) Distribution analysis of major and minor mitochondrial diameters to examine mitochondrial dimensions. The distribution analysis was performed by binning at 0.1-μm increments. The differences in diameters between F/F;− and F/F;cre mitochondria are significant according to the Kolmogorov-Smirnov (K-S) test (P < 0.001, F/F;− versus F/F;cre; D represents the value for the largest overall deviation). (J) The organization of the calcium release domains in F/F;− and F/F;cre samples is examined in further detail. The T-tubule is indicated by a black arrow and the mitochondrial surface by a white arrow. The arrowheads indicate sacks of junctional sarcoplasmic reticulum (jSR). The distance (d, shown as a dotted line) between the center of the T-tubule (shown with a circle) and the mitochondrial surface was measured, and values were averaged for the two groups (lower right).
FIG. 4.
FIG. 4.
Confocal analysis of adult cardiac myocytes indicates the presence of enlarged/spherical mitochondria. (A) F/F;− myocytes contain mitochondria with a rectangular shape that are highly ordered. (B) F/F;cre myocytes contain mitochondria with a heterogeneous morphology that can be spherical or enlarged. The scale bar is 10 μm. TMRE, tetramethylrhodamine ethyl ester. (C) Volume rendering of a z-stack collected from a region (13 by 31 μm) of an F/F;− myocyte. (D) Volume rendering of a z-stack selected from a region (13 by 31 μm) of an F/F;cre myocyte. (E, left) The mitochondrial membrane potential was estimated using dual mitochondrial labeling with JC-1. The fluorescence intensity of the aggregate form is divided by that of the monomeric form to measure the potential across the inner mitochondrial membrane (*, P was <0.05 for F/F;− versus F/F;cre mice by Student's t test [33 and 43 F/F;− or F/F;cre myocytes, respectively, from two animals per group]). (Middle) F/F;− or F/F;cre myocytes were loaded with 500 nM Mitotracker Red, and absolute units of fluorescence intensity (FI) were determined as a measure of ΔΨm. Multiple ROI were quantified from three experiments per genotype (totals of 61 and 70 ROI from 5 F/F;− and 6 F/F;cre myocytes, respectively). The laser power (561 nm) in these experiments was at 0.3%, 0.4%, and 0.4% for F/F;− myocytes and 0.5%, 0.7%, and 0.5% for F/F;cre myocytes. *, P was <0.05 for F/F;− versus F/F;cre myocytes by Student's t test. (Right) Cells loaded with 100 nM TMRM, where units of absolute (abs.) FI were determined as a measure of ΔΨm. Multiple ROI were quantified from three different experiments per genotype (total of 91 and 90 ROI from 8 F/F;− and 8 F/F;cre myocytes, respectively). The laser power (543 nm) in the respective experiments was at 1.8%, 1.8%, and 1.5% for F/F;− myocytes and at 1.5%, 1.8%, and 2.0% for F/F;cre myocytes. *, P was <0.05 for F/F;− versus F/F;cre myocytes by Student's t test. (F) The structure of the mitochondrial network is analyzed in neonatal cardiac myocytes treated with unrelated or Mfn-2-specific siRNAs (upper row) and compared with the structure of mitochondria in adult cardiac myocytes with or without Mfn-2 (lower row). The staining of mitochondria was performed with Mitotracker Red. The scale bars are 10 μm.
FIG. 5.
FIG. 5.
Functional evaluation of mitochondria in F/F;− and F/F;cre hearts. (A) Enzymatic activities in total myocardial extracts. CS, citrate synthase; IDH, isocitrate dehydrogenase; MCAD, medium-chain acyl-CoA dehydrogenase (6 and 7 preparations per group were used from 17 and 21 F/F;− and F/F;cre mice, respectively). (B and C) Organelle volume assessed in cardiac mitochondria isolated from the interfibrillar and subsarcolemmal compartments (IFM and SSM, respectively) (*, P was <0.05 for F/F;− versus F/F;cre myocytes by Student's t test; 6 and 7 preparations were used from F/F;− and F/F;cre mice, respectively). (D and E) The activity of the respiratory chain in isolated IFM and SSM mitochondria is evaluated in the presence of different substrates. These assays did not reveal statistically significant differences in state III (ADP-driven) oxygen consumption between the two genotypic groups (Student's t test was used for each substrate, with 6 or 7 preparations per genotype). P, pyruvate; M, malate; PCar, palmitoyl carnitine; S, succinate; Rot, rotenone; prot., protein; nA O, nanoatoms of oxygen.
FIG. 6.
FIG. 6.
Ca2+ retention capacity is increased in Mfn-2-depleted mitochondria. (A) Subsarcolemmal mitochondria (SSM) (upper panel) and interfibrillar mitochondria (IFM) (lower panel) were isolated from F/F;− and F/F;cre hearts and assessed for their ability to buffer extramitochondrial Ca2+. Low Ca2+ loads are sustainable by mitochondria; however, when a threshold Ca2+ load is attained, a large and abrupt increase in extramitochondrial Ca2+ that signifies mitochondrial permeability transition occurs (*, P was <0.05 by repeated-measures ANOVA, with 7 preparations per group). (B) Mitochondrial Ca2+ tolerance, defined as the Ca2+ load that is recorded during the exponential rise of extramitochondrial Ca2+, is significantly increased after genetic deletion of Mfn-2 from SSM and IFM (*, P was <0.05 by Student's t test). AU, arbitrary units.
FIG. 7.
FIG. 7.
Calcium-induced mitochondrial swelling is delayed in the absence of Mfn-2. (A) Total cardiac mitochondria were isolated from F/F;− and F/F;cre hearts and exposed to 200 μM Ca2+ to induce swelling or left untreated. The change in absorbance that occurs as a result of mitochondrial swelling is monitored over time at 520 nm and is expressed relative to the absorbance at the beginning of the assay (6 preparations per group; error bars indicate standard errors of the means [SEM]). (B) The maximum change in absorbance throughout the assay indicates that F/F;cre mitochondria are less prone to Ca2+ swelling than F/F;− mitochondria (*, P was <0.05 for untreated versus Ca2+-treated mitochondria; #, P was <0.05 for F/F;− versus F/F;cre mitochondria [6 preparations per group]). (C) The rate of mitochondrial shrinkage in response to PEG treatment is delayed in the absence of Mfn-2. Isolated mitochondria from hearts with or without Mfn-2 were preloaded with Ca2+ to induce swelling and were subsequently exposed to PEG to induce shrinkage (3 preparations per group; error bars indicate SEM). The rates of mitochondrial shrinkage in response to PEG in the absence of Ca2+ swelling were not different between the two groups (results not shown). (D) The expression levels of the different proteins responsible for MPT in hearts with or without Mfn-2. Cyp-D, cyclophilin-D; VDAC, voltage-dependent anion channel; ANT, adenine nucleotide translocase; COX-IV, cytochrome c oxidase subunit 4.
FIG. 8.
FIG. 8.
Deletion of Mfn-2 in cardiac myocytes diminishes the rate of mitochondrial depolarization in response to generation of ROS. (A) Representative image of a TMRM-loaded myocyte and time course of photostress-dependent mitochondrial depolarization. (Top) TMRM fluorescence indicates the pattern of polarized mitochondrial in myocytes. The two nuclei are seen as dark ovals. A depolarized mitochondrion (arrow) is dark. The box indicates a preselected area (30 by 35 μm) to be repetitively imaged during the photon stress experiment. (Middle) Sample images of the illuminated region at 1, 5, and 9 min. (Bottom) Image of the cell showing the permanent depolarization of mitochondria in the preselected area, while other areas of the cell contain polarized mitochondria. (B) The loss of TMRM fluorescence intensity (FI) is delayed in the absence of Mfn-2 or by pretreatment with cyclosporine (CsA). Comparison of F/F;− (purple) and F/F;cre (blue) myocytes loaded with TMRM and subjected to photon stress shows that the loss of Mfn-2 is associated with a delayed mitochondrial depolarization. Furthermore, this effect can be attributed to MPTP activation, as it is shown that CsA is able to delay the depolarization in F/F;− myocytes (orange) and can be additive to the effects associated with Mfn-2 deletion (green, dotted lines represent SEM for each group). Rel, relative. (C) Times to half-depolarization (T50) in mitochondria with or without Mfn-2 in the presence of CsA (**, P was <0.05 for untreated versus CsA-treated F/F;− myocytes; ***, P was <0.05 for untreated versus CsA-treated F/F;cre myocytes). (D) Time-dependent mitochondrial depolarization in adult myocytes in response to H2O2 exposure. Myocytes were exposed to 200 μM H2O2, and images were collected at 1-min intervals (the images shown here are pseudocolored to illustrate mitochondria). (E) Multiple areas (8 by 8 μm) were selected from 9 to 12 myocytes per group and analyzed with ImageJ for the loss of fluorescence intensity (FI) as a function of time after H2O2 treatment, and the change in fluorescence was expressed relative to the values at the beginning of the experiment. Error bars indicate standard errors of the means (21 to 27 ROI per group). (F) The FI at 1,500 s of H2O2 exposure was subtracted from the FI at 60 s to calculate the cumulative change that was expressed as a percentage of the baseline (t0) (*, P was <0.05, 21 to 27 ROI per group).
FIG. 9.
FIG. 9.
The effects of Mfn-2 on mitochondrial depolarization are context dependent. (A) Neonatal cardiac myocytes (NRCMs) were treated with unrelated (control) siRNA or with an Mfn-2-targeting siRNA and analyzed for mitochondrial depolarization in response to H2O2 exposure. (B) The loss of membrane potential is associated with increased cell death as assessed by the release of lactate dehydrogenase (LDH) into the culture medium (3 independent experiments per group; P was <0.05 for control versus Mfn-2 siRNA-treated cells after exposure to H2O2). (C) Peritoneal macrophages (Mφ) lacking Mfn-2 (F/F;creLyzM) and control macrophages (F/F;−) were loaded with TMRM and exposed to H2O2. (D) The reduction in mitochondrial depolarization as a result of Mfn-2 ablation was associated with less macrophage death and subsequent release of LDH in the medium (4 independent experiments per group; P was <0.05 for F/F;− versus F/F;creLyzM macrophages).
FIG. 10.
FIG. 10.
Mfn-2 ablation results in improved cardiac performance following ex vivo ischemia/reperfusion (I/R) injury. F/F;− and F/F;cre hearts (5 and 4 hearts, respectively) from 10-week-old mice were perfused in the Langendorff mode, subjected to 10 min of global ischemia, and followed up with reperfusion for 20 min. The systolic and developed pressures (A and B, respectively) were recorded before ischemia (baseline), during ischemia (10 to 20 min), and upon termination of ischemia (reperfusion, 20 to 40 min) (*, P was <0.05 for F/F;− versus F/F;cre hearts; **, P was <0.01 for F/F;− versus F/F;cre hearts, by repeated-measures ANOVA). This experiment was independently repeated with similar results. (C) Mitochondria from F/F;cre hearts contain higher levels of the antiapoptotic protein Bcl-2 than F/F;− hearts. Following I/R injury, mitochondria were isolated from F/F;− and F/F;cre hearts and protein extracts were analyzed. LDH, lactate dehydrogenase; Vα, complex V (FoF1 ATPase), subunit α.
FIG. 11.
FIG. 11.
Mfn-2-deficient cardiac myocytes are protected from cell death induced by hypoxia/reoxygenation and H2O2 exposure. (A) Adult cardiac myocytes were isolated from F/F;− and F/F;cre hearts and were exposed to either normoxic conditions or subjected to 1 h of hypoxia followed by 2 h of reoxygenation. At the end of the reoxygenation (or the normoxia treatment used as a control), the myocytes were stained with trypan blue to identify myocytes that had lost their membrane integrity (blue). (B) The trypan blue-positive myocytes were counted, and their number was expressed as a percentage of the total number of myocytes per field (stained and unstained). The assay indicates that loss of Mfn-2 is associated with a decreased percentage of trypan blue-positive myocytes at normoxia (*, P was <0.05 for F/F;− versus F/F;cre myocytes by one-way ANOVA) and after hypoxia/reoxygenation treatment (#, P was <0.01 for F/F;− versus F/F;cre myocytes by one-way ANOVA). The numbers of quantified fields per group are shown in circles in their respective bars, and myocytes were isolated from 6 and 5 F/F;− and F/F;cre hearts, respectively. (C) The activation of apoptosis in purified myocytes exposed to normoxia or hypoxia/reoxygenation was examined by Western blotting. The bands of cleaved (cl) caspase-9 (detected as a 39-kDa band) and the short fragment of cleaved PARP-1 (cl-PARP, detected as an ∼30-kDa band) are shown. GAPDH was used as a loading control, and the blots shown here are representative of the results of experiments performed with myocytes isolated from three hearts per group. (D) The death of purified myocytes as a result of H2O2 exposure was also examined. In this assay, myocytes under normoxic conditions were exposed to 20 μΜ Η2Ο2 for 2 h and then analyzed for trypan blue uptake. The data show that cell death is higher in myocytes expressing normal levels of Mfn-2 than in Mfn-2-depleted myocytes (#, P was <0.01 for F/F;− versus F/F;cre myocytes by one-way ANOVA). The number of quantified fields per group is shown in circles in the respective bars.
FIG. 12.
FIG. 12.
Mfn-2 deficiency in the heart results in an altered expression of outer mitochondrial membrane-associated factors and confers protection from apoptosis. (A) Western blot analysis of cardiac extracts from hearts with (F/F;−) or without (F/F;cre) Mfn-2. The expression of the proapoptotic protein Bax and the antiapoptotic protein Bcl-2 was examined. The levels of Drp-1 and Opa-1 were also assessed. (B) Heart slices taken from mice with (F/F;−) or without (F/F;cre) cardiomyocyte Mfn-2 that were subjected to 30 min of regional ischemia and 2 h of reperfusion to induce myocardial injury and cell death. The infarct areas (IA) stain negative for TTC and appear as white areas, whereas the ischemic-but-viable myocardium stains red. The nonischemic portion of the LV is stained blue. (C) Quantification of the different regions produced by LAD coronary artery occlusion was performed by planimetry and corrected for slice weight. The IA/AAR ratio provides estimation for the extent of cell death. AAR, area at risk; LV, left ventricle; IA, infarct area (*, P was <0.05 for F/F;− versus F/F;cre hearts by Student's t test [7 and 8 mice, respectively]). (D) The TUNEL assay was performed to evaluate the degree of apoptosis in response to I/R in hearts with or without Mfn-2. TUNEL-positive nuclei correspond to apoptotic cells and are stained green. The total number of nuclei was determined by DAPI (4′,6-diamidino-2-phenylindole) counterstaining (blue) and was used to calculate the ratio of apoptotic nuclei in a given field. The scale bar is 100 μm. (E) Quantification of the apoptotic nuclei after I/R in the LV of mice with or without Mfn-2. Multiple fields from two or three sections per heart were quantified for the number of green and blue nuclei, and the percentages were averaged per animal (*, P was <0.05 for F/F;− versus F/F;cre hearts by Student's t test [7 and 8 mice, respectively]).

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