© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 3705-3720 doi:10.1242/jcs.192203

RESEARCH ARTICLE Desmin and αB-crystallin interplay in the maintenance of mitochondrial homeostasis and cardiomyocyte survival Antigoni Diokmetzidou1, Elisavet Soumaka1, Ismini Kloukina1, Mary Tsikitis1, Manousos Makridakis2, Aimilia Varela3, Constantinos H. Davos3, Spiros Georgopoulos1, Vasiliki Anesti1, Antonia Vlahou2 and Yassemi Capetanaki1,*

ABSTRACT Mavroidis and Capetanaki 2002; Capetanaki et al., 2007; Psarras The association of desmin with the α-crystallin Β-chain (αΒ-crystallin; et al., 2012; Mavroidis et al., 2015). In humans, over 70 desmin encoded by CRYAB), and the fact that mutations in either one of mutations have been linked to desmin-related cardiomyopathies, of them leads to heart failure in humans and mice, suggests a potential which DCM is the most frequent (Capetanaki et al., 2015; van compensatory interplay between the two in cardioprotection. To Spaendonck-Zwarts et al., 2011). In addition, desmin aggregate αΒ formation due to caspase cleavage is an important mediator of TNF- address this hypothesis, we investigated the consequences of - α crystallin overexpression in the desmin-deficient (Des−/−) mouse -induced heart failure (Panagopoulou et al., 2008). Importantly, model, which possesses a combination of the pathologies found in regardless of the specific mutations, human hearts undergoing most cardiomyopathies, with mitochondrial defects as a hallmark. We DCM-linked failure display mitochondrial and other structural demonstrated that cardiac-specific αΒ-crystallin overexpression abnormalities, similar to those in desmin-deficient mice and mice undergoing TNF-α-induced failure. Furthermore, the R120G ameliorates all these defects and improves cardiac function to α Β α αΒ mutation in the -crystallin -chain ( B-crystallin; encoded by almost wild-type levels. Protection by -crystallin overexpression CRYAB is linked to maintenance of proper mitochondrial protein levels, ), an abundant small heat-shock protein, also causes the same inhibition of abnormal mitochondrial permeability transition pore desmin-aggregate-related DCM and heart failure with similar activation and maintenance of mitochondrial membrane potential mitochondrial defects (Wang et al., 2001; Vicart et al., 1998). Δψ αΒ Mitochondrial abnormalities seem to be a common feature of ( m). Furthermore, we found that both desmin and -crystallin are α localized at sarcoplasmic reticulum (SR)–mitochondria-associated diseases in which desmin and/or B-crystallin deficiencies are membranes (MAMs), where they interact with VDAC, Mic60 – the implicated. Previous observations suggest a physical association of core component of mitochondrial contact site and cristae organizing desmin with mitochondria, as well as an involvement of desmin in system (MICOS) complex – and ATP synthase, suggesting that these mitochondrial homeostasis (Stromer and Bendayan, 1990; Reipert associations could be crucial in mitoprotection at different levels. et al., 1999; Hnia et al., 2011), but the underlying mechanisms remain elusive. αΒ-crystallin associates with all three cytoskeletal KEY WORDS: Intermediate filaments, Small heat-shock protein, networks – microfilaments (Bennardini et al., 1992; Singh et al., Cytoskeleton, Heart failure, Mitochondria, Cristae, MAMs 2007), microtubules (Arai and Atomi, 1997; Houck and Clark, 2010) and intermediate filaments (Nicholl and Quinlan, 1994; Iwaki et al., INTRODUCTION 1989; Wisniewski and Goldman, 1998) (reviewed in Wettstein et al., Desmin, the muscle-specific intermediate filament protein, forms a 2012) – and specifically desmin (Bennardini et al., 1992; Nicholl and three-dimensional scaffold that interconnects the contractile Quinlan, 1994; Perng et al., 1999). There is also evidence that a apparatus to the nucleus, cellular organelles and the sarcolemma fraction of αΒ-crystallin is located in mitochondria (Fountoulakis (Capetanaki et al., 2007). The desmin scaffold is a primary target for et al., 2005; Martindale et al., 2005; Maloyan et al., 2005; Mitra et al., cardiomyopathy and heart failure both in mice and humans 2013); however, its role there is not completely understood. (Goldfarb et al., 1998; Capetanaki et al., 2007, 2015). Mice Mitochondria are double-membrane-bounded organelles that deficient for desmin develop both skeletal and myocardial defects are involved in important cellular processes. Proper assembly, that are strongly linked to impaired mitochondrial morphology and maintenance and function of the various mitochondrial complexes, function (Milner et al., 1996, 1999, 2000; Li et al., 1996; including oligomers of optic atrophy 1 (OPA1) (Frezza et al., Papathanasiou et al., 2015). These mitochondrial abnormalities 2006), F1F0-ATP synthase (Strauss et al., 2008) and the recently lead to cardiomyocyte death and myocardial degeneration, identified mitochondrial contact site and cristae organizing system accompanied by inflammation and fibrosis, resulting in dilated (MICOS) (Harner et al., 2011; Hoppins et al., 2011; von der cardiomyopathy (DCM) and heart failure (Milner et al., 2000; Malsburg et al., 2011) are vital to inner membrane organization, and to proper and efficient mitochondrial function. Mitofilin 1Center of Basic Research, Biomedical Research Foundation, Academy of Athens, (Mic60; also known as IMMT) (Gieffers et al., 1997; Odgren et al., Athens 11527, Greece. 2Center of Systems Biology, Biomedical Research 1996), the core component of MICOS, interacts with several inner 3 Foundation, Academy of Athens, Athens 11527, Greece. Center of Clinical, and outer mitochondrial membrane (IMM and OMM, respectively) Experimental Surgery & Translational Research, Biomedical Research Foundation, Academy of Athens, Athens 11527, Greece. proteins (von der Malsburg et al., 2011; Darshi et al., 2011; Xie et al., 2007; Harner et al., 2011; Hoppins et al., 2011; van der Laan *Author for correspondence ([email protected]) et al., 2012), thus maintaining IMM morphology and contact sites E.S., 0000-0001-8705-6741; Y.C., 0000-0002-3089-0767 between IMM and OMM, and affecting mitochondrial function, mitochondrial (mt)DNA integrity and protein and metabolite

Received 9 May 2016; Accepted 15 August 2016 trafficking. Journal of Cell Science

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To address the role of desmin and αB-crystallin, and Complete rescue of adverse remodeling in desmin-deficient their potential interplay in mitochondrial homeostasis and hearts overexpressing αΒ-crystallin cardioprotection, we investigated the consequences of αΒ- Inflammation, fibrosis and calcification are hallmarks of Des−/− crystallin overexpression in the desmin-deficient cardiomyopathy mouse myocardial degeneration and necrosis (Milner et al., 1996; model. We demonstrated that cardiac-specific αB-crystallin Mavroidis and Capetanaki, 2002; Psarras et al., 2012). Dissection of overexpression rescues desmin-deficient heart failure and that this Des−/−αΒCry4 mice revealed a complete absence of extensive takes place through maintenance of proper mitochondrial calcification in the myocardium. Further histological analysis composition, structure and function, through inhibition of revealed no inflammatory infiltration and a minimal amount of abnormal mitochondrial permeability transition pore (mPTP) fibrosis in the ventricular walls (Fig. 2A–D). Quantification of activation and through cardiomyocyte death prevention. histological section images confirmed the significant reduction of Moreover, we found that among other sites, both desmin and αΒ- fibrosis in the Des−/−αΒCry myocardium (Fig. 2C). Comparison of crystallin localize at sarcoplasmic reticulum (SR)–mitochondria- the two transgenic lines Des−/−αΒCry4 and Des−/−αΒCry6 associated membranes (MAMs), where they interact with VDAC. revealed that a 5.4-fold overexpression of αB-crystallin was more Furthermore, we have unraveled a potential link of desmin and αB- beneficial when compared to a 2.6-fold overexpression. Therefore, crystallin with mitochondrial morphology and bioenergetics the subsequent studies were done using line Des−/−αΒCry4, through their interaction with Mic60 and ATP synthase. designated from now on as Des−/−αΒCry. Evans-Blue dye (EBD) analysis was performed to detect cellular RESULTS membrane permeability. Wild-type cardiomyocytes were Generation of Des−/− mice overexpressing αΒ-crystallin in the impermeable to EBD because their membranes were intact − − heart (Fig. 2E,F). In contrast, Des / hearts appeared to have In order to examine the effect of αΒ-crystallin overexpression in the considerably large areas of EBD-positive cells, especially in Des−/− myocardium, we generated transgenic mice overexpressing regions with inflammatory infiltration. In Des−/−αBCry αΒ-crystallin specifically in the heart under the control of the myocardium there were no EBD-positive cardiomyocytes, leading α-myosin heavy chain (αMHC) promoter. Among different lines to the conclusion that αB-crystallin overexpression protects Des−/− generated, two were chosen for further investigation, namely myocardium from necrotic cell death. αΒCry4 and αΒCry6, with high and low αB-crystallin expression levels respectively (Fig. 1A; Fig. S1A). These mice were crossed αΒ-crystallin overexpression considerably improves cardiac − − − − with Des / mice to generate αΒ-crystallin overexpressing Des / function and results in 100% survival during obligatory − − mice (Des / αΒCry). Quantification of total αB-crystallin protein swimming exercise levels from hearts of wild-type, Des−/− and transgenic lines revealed Heart failure development with aging is characteristic of Des−/− 1.3-fold higher αB-crystallin levels in Des−/− mice, a 5.4-fold hearts. To explore the effect of αΒ-crystallin overexpression on increase in line αΒCry4 and a 2.6-fold increase in line αΒCry6 in cardiac function, we performed standard morphometric analysis and comparison to wild type. two-dimensional directed M-mode echocardiography (Fig. 3; Table S1). Des−/−αBCry mice showed a significant improvement − − Overexpression of αΒ-crystallin rescues its compromised in all parameters tested in comparison to Des / mice. The − − Z-disc localization and allows for more efficient improvement in left ventricular function of Des / αBCry mice was mitochondrial localization in Des−/− hearts very extensive, reaching almost wild-type levels (96% of the wild- It has been shown that, upon stress, αΒ-crystallin translocates from type fractional shortening; Fig. 3Bc), consistent with the a cytosolic pool to Z-discs (Golenhofen et al., 1998), where it improvement in left-ventricular-end systolic diameter, which was colocalizes with desmin and α-actinin. Immunofluorescence also comparable to wild-type levels (Fig. 3Bb). Posterior wall microscopy was performed to determine whether desmin deficiency thickness was also increased by 35% in the Des−/−αBCry compared affects the Z-disc localization of αΒ-crystallin. We found that, in the to the Des−/− group, consistent with the absence of degeneration in unstressed wild-type myocardium, αB-crystallin colocalized only these hearts (Fig. 3Bd). Finally, the ratio of left ventricular radius to partially with desmin and α-actinin at the Z-disc, and was also located posterior wall thickness, an indicator of left ventricular wall stress, at the intercalated discs (Fig. S1B). This localization pattern was was significantly lower in Des−/−αBCry mice compared to that in enhanced under stress as a result of the mice swimming (Fig. 1D). In Des−/− mice (Fig. 3Be). the absence of desmin, the Z-disc localization of αB-crystallin was In order to investigate whether this cardioprotection also occurs mostly lost, but its localization at intercalated discs was retained under stress conditions, wild-type, Des−/− and Des−/−αΒCry mice (Fig. 1D), possibly owing to other potential binding partners at the were subjected to an obligatory swimming exercise protocol for intercalated discs. Overexpression of αB-crystallin in the Des−/− 24 days. The Des−/− mice had a considerably reduced ability to αBCry4 myocardium rescued its Z-disc localization (Fig. 1D), and handle stress because 50% of them died during swimming more importantly, a large amount of αB-crystallin was located in (in comparison to 0% of wild type) (Fig. 3C). The survival rate of mitochondria (Fig. 1Bb). Mitochondrial localization of αB-crystallin Des−/−αΒCry mice was 100%, strongly demonstrating the powerful also occurred in wild-type cardiomyocytes but could not be easily cardioprotective action of αΒ-crystallin overexpression against detected. Under the stress of swimming, the total protein levels stress. of both desmin and αB-crystallin were increased (Fig. 1Ba,Ca,Cb), and this allowed for easier detection of αB-crystallin in the Rescue of Des−/− cardiomyocyte ultrastructural defects by − − mitochondrial fraction in both wild-type and Des / myocardium αΒ-crystallin overexpression (Fig. 1Cc). This was more obvious in the corresponding αB- In an effort to understand the mechanism by which the crystallin-overexpressing myocardium [wild-type (wt)αBCry4 and overexpression of αΒ-crystallin protects Des−/− myocardium Des−/−αBCry4]; however, the ratio of mitochondrial to cytosolic against degeneration and improves cardiac function, we initially −/− levels remained the same. examined its effects on cardiomyocyte ultrastructure. Des Journal of Cell Science

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Fig. 1. Increased αΒ-crystallin levels overcome its desmin-dependent stress-induced relocation. (A) Expression levels of αB-crystallin protein in the heart − − from 2-month-old wild-type (WT), Des / and αΒ-crystallin-overexpressing (des−/−αBCry4 and des−/−αBCry6) transgenic mice. GAPDH, loading control. (B) Western blot analysis of total heart lysates (a) and subcellular fractions (b) from mice in control conditions and after a swimming exercise reveals increased − − levels of αΒ-crystallin (αΒCry) in the mitochondria of Des / αBCry4 hearts; GAPDH, cytosolic marker; cytochrome c oxidase IV (COXIV), mitochondrial marker. The arrowhead in Bb indicates nonspecific bands. (C) Cardiac protein levels of (a) desmin and (b) αB-crystallin under normal and stressed (swimming) conditions, and (c) the protein levels of αB-crystallin located in the cytosol (cyt) and mitochondria (mit). Error bars show mean±s.e.m. (a) ***P<0.001 vs wild-type control, − − − − *P≤0.05 vs wtαBCry control; (b) **P<0.001 vs wild-type control, ***P<0.0001 vs Des / control and swimming, *P<0.05 vs Des / αBCry control (Student’s unpaired t-test). (D) Double immunofluorescence staining for αΒ-crystallin and α-actinin in frozen myocardial tissue sections from 6-month-old mice (des −/−αBCry4), fixed right after swimming (blue staining of nuclei, DAPI; arrows, intercalated discs). Scale bars: 10 μm. Journal of Cell Science

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− − Fig. 2. Complete amelioration of adverse remodeling in Des / hearts overexpressing αΒ-crystallin. (A) Histological Masson’s trichrome staining of heart − − − − − − sections from 3.5-month-old wild-type (wt; n=7), Des / (n=7), Des / αBCry4 (n=10) and Des / αBCry6 (n=7) mice. Fibrosis was detected by blue staining of − − − − collagen deposition. (B) Hematoxylin and eosin staining of 23-day-old wild-type (n=6), Des / (n=12), Des / αΒCry (n=15) and wtαΒCry (n=6) mice, showing the − − inflammatory infiltration in the Des / myocardium compared to that in the other genotypes. Scale bars: 20 µm. (C) Semi-quantitative determination of myocardial − − − − − − fibrosis using ImageJ software analysis. **P<0.01 vs wild-type and Des / αBCry6; ***P<0.0001 vs Des / ; #P<0.001 vs wild type and Des / αBCry4. (D) Assessment of cardiac inflammation using 23-day-old hearts and a conventional grading system (see Psarras et al., 2012). ***P<0.0001 vs wild type, − − Des / αΒCry and wtαΒCry. (E) Evaluation of cardiomyocyte membrane permeability by Evan’s-Blue (red) and α-actinin (green) staining of frozen heart sections from 23-day-old mice; blue staining of nuclei, DAPI. Scale bars: 100 µm. (F) A quantification of the EBD-positive area by using ImageJ software analysis. ***P<0.0001. In the box-and-whisker plots in D and F, the boxes indicate the interquartile range of the samples, the lines represent the median values and the ’ whiskers represent the outliers. All error bars show mean±s.e.m. (unpaired Student s t-test). Journal of Cell Science

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− − Fig. 3. αΒ-crystallin overexpression considerably improves cardiac function, survival rate during obligatory exercise and Des / cardiomyocyte − − ultrastructural defects. Mice at 7 months of age were subjected to echocardiography. Des / αBCry mice showed a significant improvement in left ventricular (LV) function reaching a fractional shortening (FS) value that was 96% of that of wild type (wt). (A) Representative M-mode echocardiograms from each mouse genotype. (B) Group data (mean±s.e.m) for left ventricular end diastolic diameter (a, LVEDD), end systolic diameter (b, ESD), FS (c), posterior wall dimension − − (d, PWd), ratio of left ventricular radius to PWd (e, r/h), ejection fraction (f, EF); *P<0.01, **P<0.001 and ***P<0.0001 vs Des / (ANOVA with Bonferroni–Dunn − − − − post-hoc test). (C) Survival curve of 3-month-old wild-type (n=12), Des / (n=12) and Des / αBCry (n=13) mice during a swimming protocol. The error bars − − − − correspond to mean±s.e.m.; P<0.0001 Des / vs wild type and Des / αBCry at all time points (unpaired Student’s t-test). (D) Myocardial tissue ultra-structure from 3-month-old mice that were examined by using transmission electron microscopy. Scale bar: 2 µm. (E) Higher magnification images of the myocardial − − mitochondria. Desmin deficiency affects the ultrastructure of the IMM, leading to excessive disorganization of cristae. The mitochondria from Des / αΒCry transgenic myocardium appear normal. m, mitochondria; N, nucleus; c, cristae. Scale bars: 0.2 µm. Journal of Cell Science

3709 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 3705-3720 doi:10.1242/jcs.192203 cardiomyocytes are characterized by various early-appearing address this possibility, we incubated adult cardiomyocytes with mitochondrial defects (Milner et al., 2000), followed by other tetramethylrhodamine methyl ester (TMRM), a potentiometric structural abnormalities and loss of muscle integrity (Fig. 3D). indicator that is targeted to mitochondria. The TMRM intensity was Mitochondrial abnormalities include disturbed shape and 21% lower in Des−/− cardiomyocytes compared to that in wild type, positioning, fragmentation, aggregation, swelling of the indicating reduced Δψm (Fig. 4D,E). Time-lapse confocal scanning mitochondrial matrix, disruption and fragmentation of the cristae showed a more rapid loss of TMRM in Des−/− mitochondria relative and degeneration (Fig. 3E). In contrast, none of these ultrastructural to that in wild-type mitochondria, consistent with mitochondrial defects are present in Des−/−αΒCry mice. Myofibrils were more membrane depolarization (Fig. 4F; Fig. S2). αB-crystallin properly aligned and mitochondria, located parallel to myofibrils, overexpression in Des−/− myocardium significantly increased appeared normal, without signs of fragmentation, cristae both the intensity and retention time of TMRM. To examine abnormalities or destruction. These data suggest that mitochondria whether the accelerated TMRM loss in Des−/− cardiomyocytes was are an important target of αΒ-crystallin cytoprotection. due to mPTP activation, we treated cardiomyocytes with CsA, significantly attenuating TMRM loss. αΒ-crystallin overexpression provides anti-oxidant protection to Des−/− cardiomyocytes αΒ-crystallin overexpression restores changes in the levels To further investigate the mechanism by which αΒ-crystallin protects of important mitochondrial proteins caused by desmin against mitochondrial defects, we analyzed its effects on adult deficiency cardiomyocyte reactive oxygen species (ROS) levels using the In an effort to understand the underlying mechanisms responsible −/− molecular probe CM-H2DCFDA (Fig. 4A,B). Des for both the observed mitochondrial defects in the absence of cardiomyocytes showed a significantly higher degree of ROS over desmin and, most importantly, the unprecedented extensive rescue those in the wild-type, whereas Des−/−αΒCry cardiomyocytes were of these defects by αΒ-crystallin overexpression, we conducted characterized by a lower degree of ROS. Under oxidative stress proteomic analysis to identify any crucial alterations in the conditions, simulated by incubation with H2O2,therewasa mitochondrial proteome of the different genotypes. The results significant decrease of ROS levels in both Des−/−αΒCry and revealed diminished levels of enzymes involved in the citric acid wtαBCry compared to Des−/− and wild-type cells respectively, cycle, lipid metabolism and fatty acid β-oxidation in the Des−/− indicative of the protection provided by αΒ-crystallin overexpression. compared to wild-type mitochondria (Table 1; Table S3, Fig. S3). To further investigate the mechanism of anti-oxidant protection by Moreover, in Des−/− mitochondria, the levels of many subunits and αΒ-crystallin, we measured the levels of glutathione (GSH). In Des−/ components of the electron transport chain were decreased, along − hearts, glutathione levels are decreased by 33% and the GSH: with components of complexes linking different metabolic glutathione-disulfide (GSSG) ratio by almost 50% in comparison to pathways. The most extensive change was found with the cristae wild type (Fig. 4C). Overexpression of αΒ-crystallin increased junction protein Mic60, the levels of which were reduced by 78% glutathione and GSH:GSSG levels to 87% and 95%, respectively, of compared to those in wild type (Table 1, Fig. 5A,B). On the those in wild type, suggesting that, at least partially, the mechanism of contrary, in the mitochondria of Des−/−αΒCry myocardium, most of anti-oxidant protection by αΒ-crystallin occurs through an increase of the proteins that showed decreased levels in Des−/− mice, including reduced glutathione levels. Mic60 protein, were apparently restored (Table 1, Fig 5; Table S4). In agreement with the western blotting data (Fig. 1B), proteomic αΒ-crystallin overexpression inhibits abnormal activation of analysis revealed that much higher levels of αΒ-crystallin localize to − − the mitochondrial permeability transition pore and the the mitochondria of the Des / αΒCry hearts in comparison to those − − dissipation of mitochondrial membrane potential of Des / hearts. The levels of some of the differentially expressed Mitochondrial swelling and increased ROS levels have been linked proteins were also evaluated by western blotting (Fig. 5A,B). In to abnormal activation of the mPTP. Therefore, we investigated addition, we investigated the mitochondrial levels of adenine whether the above demonstrated mitoprotection and nucleotide translocator (ANT), an IMM protein with known cardioprotection by αΒ-crystallin overexpression takes place mitochondrial and cardiomyocyte function (Narula et al., 2011; through inhibition of mPTP activation. First, we determined the Vogelpohl et al., 2011) that cannot be easily detected by proteomic sensitivity of isolated mitochondria to increased Ca2+ levels in the analysis. This analysis revealed that, in contrast to total ANT levels, presence or absence of the mPTP inhibitor cyclosporin A (CsA). As mitochondrial ANT levels were reduced in Des−/− myocardium, a expected, Des−/− mitochondria swelled more than wild-type defect ameliorated by αB-crystallin overexpression (Fig. 5A,B). mitochondria at the same Ca2+ concentration, as measured by the − decrease in absorbance at 540 nm (Table S2). By contrast, Des / Desmin and αΒ-crystallin interact with proteins that are − αBCry mitochondria showed a significant decrease in swelling, important for cristae morphology indicating that overexpression of αB-crystallin improves the As demonstrated above, one of the most interesting proteins diminished capacity of Des−/− mitochondria to resist exposure to downregulated in Des−/− mitochondria is Mic60 (Table 1, Fig. 5A), Ca2+. Addition of CsA before the Ca2+ challenge significantly consistent with the shown cristae morphology defects (Fig. 3D,E). abrogated mitochondrial swelling. This finding demonstrates that Given that both these defects are rescued by αΒ-crystallin first, the reduced ability of Des−/− mitochondria to resist increased overexpression, we aimed to further unravel the underlying Ca2+ levels (Weisleder et al., 2004) is mPTP dependent and second, mechanism. We used GST-tagged desmin and co- the mechanism of mitoprotection and inhibition of necrotic cell immunoprecipitation to examine whether desmin interacts with death of Des−/− myocardium by αB-crystallin overexpression Mic60, and therefore, whether loss of this interaction in Des−/− involves suppression of mPTP activation. cardiomyocytes affects its proper mitochondrial targeting and/or In the Des−/− heart, mitochondrial abnormalities, including stabilization. Indeed, we found that desmin interacts with Mic60, swelling, can lead to dissipation of mitochondrial membrane and as expected, with αΒ-crystallin (Fig. 5C,D). More importantly, potential (Δψm), a critical event in the process of cell death. To we demonstrated that αΒ-crystallin also associates with Mic60 Journal of Cell Science

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− − Fig. 4. αΒ-crystallin overexpression provides anti-oxidant protection to Des / cardiomyocytes, inhibits abnormal activation of the mPTP and the Δψ dissipation of m. (A) Staining of live adult cardiomyocytes in culture with CM-H2DCFDA to detect ROS, in control conditions and after challenging with H2O2. μ Color graduation from light blue to brown corresponds to the intensity of CM-H2DCFDA fluorescence and consequently the level of ROS. Scale bar: 250 m. (Ba) Semi-quantitative determination of CM-H2DCFDA intensity. Values are normalized to those of wild type (wt) (mean±s.e.m.), n=9; **P<0.01 vs wild type, −/− ’ ≤ *P<0.05 vs Des (unpaired Student s t-test). (b) Same as panel Ba after incubation with H2O2; ***P<0.001 and #P 0.05 vs wild type, **P<0.01 and *P<0.05 vs − − − − Des / ; n=9. (C) Total glutathione levels (a) and GSH:GSSG ratio (b) in heart tissue homogenates from 6-month-old mice. Des / heart displays only 67%±4.7 (mean±s.e.m.) of the total GSH and 51.43%±11.10 of the reduced GSH compared to wild-type heart, wheteas αΒCry overexpression increases these levels − − up to 87%±9.3 and 95.39%±12.38, respectively. (a)***P≤0.001 vs the other genotypes, #P<0.001 vs wild type; (b) ***P≤0.001 vs wild type, *P≤0.05 vs Des / ; ’ Δψ n=4 (unpaired Student s t-test). (D) Determination of mitochondrial m by labeling adult cardiomyocytes with the potentiometric probe TMRM. (E) Semi-quantitative determination of the initial TMRM fluorescence; *P<0.05 vs the other genotypes, #P≤0.05 vs untreated (unpaired Student’s t-test). (F) Loss Δψ of mitochondrial m analysis using 20-min time-lapse confocal imaging of cardiomyocytes loaded with TMRM probe (see also Fig. S2). The mPTP inhibitor − − cyclosporin A (CsA) was used to confirm the mPTP activation. The error bars show mean±s.e.m.; P<0.01 vs Des / and CsA; n=9.

(Fig. 5D), both in wild-type and Des−/− myocardial extracts, in the case of desmin), we found that the β subunit of the F1 catalytic strongly suggesting that Mic60 is one of the αΒ-crystallin targets in domain of ATP synthase, another protein important for determining the Des−/− mitochondria. Using similar immunoprecipitation cristae structure (Paumard et al., 2002) and mitochondrial function studies, (and immunoprecipitation coupled to proteomic analysis (Zick et al., 2009), interacts with desmin as well as with Journal of Cell Science

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Table 1. αB-Crystallin overexpression restores several mitochondrial remain intact. The digestion pattern of desmin and αB-crystallin − − enzymes and proteins that are diminished in Des / hearts resembled that of mitofusin 2, an OMM protein, as detected by an − − − − Accession Des / vs Des / αBCry antibody that recognizes a part of mitofusin 2 that is exposed to the − − Protein name name (mouse) wild type vs Des / cytosol. In contrast, overexpressed αB-crystallin appears to localize α-crystallin B-chain CRYAB – 66 inside mitochondria (Fig. 6Ab). Mic60 (mitochondrial inner IMMT 0.22 3.6 Next, we performed subcellular fractionation of rat and mouse membrane protein) hearts to further determine the localization of desmin. Interestingly, Dihydrolipoyl dehydrogenase DLDH 0.47 3.7 although we found desmin in the SR fraction, it was mainly enriched 3-mercaptopyruvate THTM – 3.6 in the SR–MAMs (Fig. 6B). In contrast, αB-crystallin was found sulfurtransferase enriched more in the SR fraction and less so in the MAMs and pure Pyruvate dehydrogenase E1 ODPB 2.3 3.4 α component subunit β mitochondrial fraction. Furthermore, overexpressed B-crystallin 2-oxoglutarate dehydrogenase ODO1 – 3.2 was also enriched in the SR fraction but even more so in the MAMs Succinyl-CoA ligase (ADP- SUCB1 – 3.5 and in pure mitochondria (Fig. 6C). The localization of desmin was forming) subunit beta confirmed by using immunogold labeling studies (Fig. 6D; Fig. S4). Trifunctional enzyme subunit α ECHA 0.36 2.6 In addition to the expected places, such as intercalated discs Succinate dehydrogenase DHSA 0.54 3.7 (Fig. S4) and Z-lines, desmin was found to associate with (ubiquinone) flavoprotein mitochondria and membranous structures, most likely SR, in subunit NADH dehydrogenase NDUS2 0.69 2.3 close proximity to mitochondria. (ubiquinone) iron-sulfur A previous yeast two-hybrid screening of a heart cDNA library protein 2 using the entire desmin molecule as bait, conducted in our NADH dehydrogenase NDUAA – 2 laboratory, had shown binding of desmin to VDAC (our (ubiquinone) 1-alpha unpublished results). In addition to its mitochondrial localization, subcomplex subunit 10 VDAC is also enriched in MAMs, and furthermore it has been Electron transfer flavoprotein- ETFD 0.58 2.1 ubiquinone oxidoreductase found to interact with Mic60 (Hoppins et al., 2011). Therefore, we NADH dehydrogenase NDUB9 0.50 – reconfirmed the binding of VDAC to desmin using both co- (ubiquinone) 1-beta immunoprecipitation studies as well as GST pulldown assays subcomplex subunit 9 (Fig. 7A,B). To determine the corresponding binding regions of NADH dehydrogenase NDUV1 0.29 – VDAC and desmin, we generated several deletion constructs and (ubiquinone) flavoprotein 1 used them in reciprocal GST pulldown experiments. As The differentially expressed proteins in purified cardiac mitochondria extracts demonstrated in Fig. 7D, the N-terminal domain of VDAC is from 3-month-old mice are given as the fold change relative to wild-type levels − − − − necessary for its binding to desmin, whereas the C-terminal tail in the case of Des / mitochondria and as the fold change relative to Des / − − domain of desmin and, to a lesser extent, the rod domain, are levels in the case of Des / αBCry mitochondria. Samples were analyzed by using two-dimensional gel electrophoresis coupled to mass spectrometry sufficient for association of desmin with VDAC. The finding of (n=4), and differentially expressed protein spots were identified using MALDI- desmin–VDAC binding was further strengthened by double TOF–TOF-MS. All the identified proteins are provided in Tables S3 and S4. immunogold labeling studies, which confirmed the close proximity of these two proteins in vivo (Fig. 6D; Fig. S4). αB- αΒ-crystallin (Fig. 5C,D). Loss of this interaction in the absence of crystallin was also co-immunoprecipitated with VDAC, consistent desmin, combined with the disrupted cristae and depolarized Δψm, with earlier reports (Mitra et al., 2013). The demonstrated is expected to impact the ATP content, which we indeed confirmed associations of desmin and αB-crystallin with VDAC, Mic60 and to be 40% lower in the Des−/− cardiomyocytes compared to that in ATP synthase could somehow facilitate the formation and/or wild type (Fig. 5E). αΒ-crystallin overexpression increased this ATP stabilization of a scaffold-like super complex extending from SR– content to 90% of the wild-type levels. mitochondria contact sites to MICOS and ATP complexes. These findings support the view that the loss of desmin association with these proteins could be a key mediator of pathologies resulting αB-crystallin restores the formation of Mic60-containing from loss of desmin. Given that αΒ-crystallin also associates with macromolecular complexes that are affected by desmin − − these proteins, its higher levels in the Des / αΒCry cardiomyocytes, deficiency in comparison to its basal levels in Des−/− cells, might be able to We speculated that desmin and αB-crystallin association with Mic60 compensate for the impact of desmin loss on Mic60 and ATP could affect the formation or stabilization of Mic60-containing synthase function. complexes. We analyzed crude mitochondria from mouse heart that had been solubilized with digitonin by using blue native PAGE in the Desmin and αB-crystallin are located at the SR and MAMs, first dimension and SDS-PAGE in the second dimension, followed by where they associate with VDAC detection of Mic60 by western blotting (Fig. 7E,F). In the absence of The question raised by the amelioration of Des−/− mitochondrial desmin, the number of the protein complexes containing Mic60 was pathology by αB-crystallin overexpression is how can desmin and much lower compared to those in wild type, consistent with the αB-crystallin, two mainly cytoplasmic proteins, interact with IMM diminished Mic60 protein levels found in Des−/− mitochondria proteins and impact mitochondrial homeostasis. To address this (Table 1, Fig. 5A). Importantly, as determined by using much lower question, we first performed protease accessibility assays to protein levels, the higher-molecular-mass complexes of Mic60 were examine whether a fraction of desmin and αB-crystallin is located proportionally less than the corresponding lower-molecular-mass inside the mitochondria. Crude mitochondria from wild-type hearts complexes, indicating a possible facilitating role of desmin in the were isolated and digested with proteinase K. As shown in Fig. 6A, formation or stabilization of these complexes (Fig. 7F; Fig. S4). All both desmin and αB-crystallin were degraded by proteinase K, these data are consistent with the cristae defects of Des−/− mitochondria whereas the IMM protein ANT and the OMM protein VDAC (Fig. 3). Overexpression of αB-crystallin in the absence of desmin Journal of Cell Science

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Fig. 5. Desmin and αB-crystallin bind to Mic60 and ATP synthase β. (A) Western blot analysis of total and mitochondrial fractions isolated from hearts of the indicated genotypes. Mic60 is expressed as two isoforms of 88 and 90 kDa. Wt, wild type. (B) Densitometry analysis of mitochondrial (mit) protein levels. COXIV − − − − was used as loading control. Plots represent the mean±s.e.m. of more than five experiments; *P≤0.05 vs Des / ,***P≤0.001 vs Des / , #P<0.05 vs wild type, ^P<0.001 vs wild type (unpaired Student’s t-test). (C) GST pulldown assay using wild-type mouse heart mitochondrial extract. Arrowhead, nonspecific binding of 55-KDa IgGs. (D) Co-immunoprecipitation (CO-IP) analysis using cardiac mitochondrial extracts. Arrowheads, nonspecific binding of 55- and 25-kDa IgGs. (E) ATP − − content of adult cardiomyocytes normalized to wild type (mean±s.e.m, n=8); ***P≤0.0001 vs wild type, *P<0.05 vs Des / (unpaired Student’s t-test). restored the abundance of all the different-sized Mic60 complexes to swimming stress conditions, we obtained similar results with only a their normal levels. Importantly, desmin and αB-crystallin appeared to slight shift in the molecular mass of VDAC complexes, as observed in be in the same complexes as Mic60 and VDAC, supporting the idea of the Des−/− mitochondria (Fig. 7F). This potentially reflects a partial a functional role in the interface of SR with OMMs and IMMs. Under dissociation of Mic60 and VDAC complexes. Journal of Cell Science

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− − Fig. 6. Desmin and αB-crystallin are located at the SR and MAMs. (A) Cardiac mitochondria (mit) from wild-type (a) and Des / αBCry (b) mice were digested with proteinase K (prot K) under the indicated conditions. VDAC and mitofusin 2 (Mfn2) were used as OMM markers, whereas ANT was used as an IMM marker. Osmotic, 20 mM KCl. (B) Subcellular fractions from wild-type hearts were analyzed by western blotting for known markers of cytosol (α-actinin), mitochondria (MnSOD), MAMs and light membranes (LM; mostly containing SR, distinguished by VDAC, Mfn2, RYR). wt, wild type. (C) αB-crystallin subcellular localization in − − − − wild type (1), Des / (2) and Des / αBCry (3) hearts. (D) Electron micrographs of double immunogold labeling of desmin (10-nm particle) and VDAC (25-nm particle). Desmin is found at Z-discs (z, black arrows), as expected (a), and in intimate association with mitochondria (m, black arrowheads) (a–c′) and membrane structures attaching mitochondria, most possibly SR and MAMs (white arrows) (c, c′), where it colocalizes with VDAC (white arrowheads) (a, a′,b,b′). a′–c′ are higher magnification images of the boxed areas. Scale bars: 0.1 μm.

DISCUSSION overexpression definitively provides extensive mitoprotection to − − − − Mitoprotection as an important mechanism of αΒ-crystallin- Des / cardiomyocytes. The earliest features of the Des / mediated cardioprotection in desmin-deficient heart failure pathology are mitochondrial structural perturbations, evident as We have demonstrated that αB-crystallin overexpression in the early as the second week after birth, before any other cardiac Des−/− myocardium completely halts the development of heart dysfunction arises (Milner et al., 2000). These findings are further failure. The present results show that, although the mechanisms of supported by additional studies showing that overexpression of cardioprotection might be occurring at various levels, αB-crystallin Bcl-2, which has a known protective action on mitochondria, Journal of Cell Science

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Fig. 7. Desmin and αB-crystallin are found in the same super-complex with Mic60 and VDAC. GST pulldown assay (A) and VDAC immunoprecipitation analysis (B) using cardiac mitochondrial (mit) lysates. Arrowheads, nonspecific binding of 25-kDa IgGs. BSA, bovine serum albumin; wt, wild type. (C) Schematic representation of desmin (a) and VDAC (b), and their deletion mutants. Des, full-length desmin; H, head; HR, head-rod; R, rod; RT, rod-tail; and T, tail domains. VDAC, full-length VDAC; ΔNterm, deletion of the α-helix N-terminus of VDAC; Δβ1β4, deletion of the first four β-strands. (D) GST pulldown analysis of desmin − − and VDAC domain interactions using mitochondrial lysates from wild-type and, in the case of VDAC, Des / hearts. (E) Heart mitochondria, solubilized with digitonin, were analyzed by blue native (BN) PAGE in the first dimension. The Coomassie-Blue dye contained in the loading buffer allows the visualization of the proteins, which serves as a loading control. (F) Analysis of solubilized mitochondria from animals in control conditions and after swimming exercise in the second dimension by using SDS-PAGE analysis. Red box, macromolecular complexes of Mic60 diminished in the absence of desmin and were restored by

αB-crystallin overexpression; blue box, shift of Mic60 and VDAC macromolecular complexes to a lower molecular mass after swimming stress. Journal of Cell Science

3715 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 3705-3720 doi:10.1242/jcs.192203 provides significant improvement of Des−/− cardiomyopathy located at the contact sites (Harner et al., 2011) and has recently (Weisleder et al., 2004). Our studies demonstrated an increase in been implicated in mitochondrial protein import (van der Laan oxidative stress, an increased activation of mPTP and a decreased et al., 2012). It is thus conceivable that the decrease of Mic60 in the Δψm in the absence of desmin. All these mitochondrial defects are absence of desmin could compromise protein import into corrected in the Des−/−αBCry myocardium, in which mitochondria mitochondria. Our proteomic analysis revealed that the levels of have an apparently wild-type phenotype. It has been shown that the many enzymes involved in aerobic respiration are diminished in the αB-crystallin mutation R120G, known to cause desminopathy Des−/− cardiomyocytes (Table 1; Tables S3, S4), whereas αΒ- characterized by desmin and αB-crystallin aggregation, also leads to crystallin overexpression restores them, thus conferring to the cell redox imbalance (Rajasekaran et al., 2007). Defining the extent to high-energy electrons in the form of NADH and FADH2, which can which these aggregates contribute to the development and be used for ATP generation by oxidative phosphorylation. progression of the disease is of great importance. Clearing away In addition to Mic60, F1F0-ATP synthase affects cristae the aggregates improves this model of proteotoxicity (Li et al., 2011; membrane structure by imposing curvature through dimerization Su et al., 2015; Cabet et al., 2015; Pattison et al., 2011; Gupta et al., and oligomerization (Strauss et al., 2008; Dudkina et al., 2006; 2014), but it does not compensate for the loss of function of the Minauro-Sanmiguel et al., 2005), thus providing the required aggregated proteins. Previous and present data with the Des−/− conditions to enhance the catalytic capacity of the oxidative model – which shows no desmin and αB-crystallin aggregates but phosphorylation system (Strauss et al., 2008). Recently, displays similar cellular and functional defects with those of mitochondrial cristae shape has been found to determine desmin- and αB-crystallin-related cardiomyopathy (Milner et al., respiratory chain super-complex assembly and respiratory 1996, 1999, 2000; Li et al., 1996; Pattison et al., 2011; Wang et al., efficiency (Cogliati et al., 2013). Such efficiency minimizes the 2003; Wang and Robbins, 2006; McLendon and Robbins, 2011) – downstream ROS production discussed above, consistent with the suggest alternative causes for this pathology, in addition to the gain proposed upstream action of αΒ-crystallin. All these data are also of toxic function (Bhuiyan et al., 2013). consistent with the significant decrease of ATP levels in Des−/− hearts and their restoration by αΒ-crystallin overexpression. αB-crystallin and desmin – old partners in a new game Moreover, recent work indicates that the mPTP can be formed The association of desmin with αΒ-crystallin and the fact from ATP synthase dimers (Giorgio et al., 2013). The interaction of that mutations in either one of them lead to heart failure with desmin and αΒ-crystallin with both ATP synthase and VDAC common characteristics suggest a potential compensatory interplay suggests a possible role of these proteins in the regulation of between these two proteins in mitoprotection and consequently mitochondrial bioenergetics and mPTP opening. cardioprotection. Indeed, we found that both proteins are protecting The present data suggest that in normal heart the desmin mitochondria by, among other possible mechanisms, maintaining cytoskeletal network could facilitate the mitoprotective and proper composition of mitochondrial proteins. This could take cytoprotective roles of the endogenous αΒ-crystallin. In the place either though facilitation of efficient protein targeting and absence of desmin, αB-crystallin loses its Z-disc localization and transport of the nucleus-encoded mitochondrial proteins into the thus the ability to reach the required high concentrations at a Z-disc- mitochondria, or through the support of the formation and/or proximal SR– or endoplasmic reticulum (ER)–mitochondrial sites stabilization of mitochondrial protein complexes. Up to now, the for its proper function. Indeed, much higher levels of αB-crystallin former possibility was considered more probable for a desmin- are required to facilitate the same level of protection in the absence based scaffold, which, although it does associate with mitochondria of desmin. This is consistent with the presently proposed through plectin (Winter et al., 2008), is mainly cytosolic. The involvement of both proteins in the ER–mitochondria organizing present study identifies a new way by which desmin can regulate network, which might constitute only part of the function of these mitochondrial homeostasis. The contact sites where SR links to the partners in mitochondrial behavior. OMMs and IMMs are established sites for multiple cellular processes, including Ca2+ and metabolite transfer, lipid MATERIALS AND METHODS metabolism, mitochondrial shape regulation, and autophagosome Transgenic animal generation Des−/− and inflammasome formation (Naon and Scorrano, 2014). The mice had been previously generated in the 129SV inbred background (Milner et al., 1996). Transgenic mice overexpressing αΒ- desmin scaffold, located at these contact sites, could contribute to all α – crystallin were generated in the C57BL/6J background, using the MHC of the above processes, and particularly to the maintenance of SR promoter (Genbank ID U71441) (Subramaniam et al., 1991), driving mitochondria proximity. This proximity allows the desmin scaffold, expression of full-length murine αΒ-crystallin cDNA, followed by the together with αΒ-crystallin, to facilitate directed protein and bovine growth hormone poly-A sequence and backcrossed onto a pure metabolite targeting to mitochondria. As shown here, and as 129SV mouse strain for several generations. The animals were used previously suggested by others (Fountoulakis et al., 2005; independent of sex. The approved procedures for animal care and treatment Martindale et al., 2005; Maloyan et al., 2005), αB-crystallin does were followed, according to institutional guidelines following those of the localize in and outside of mitochondria, and thus, could support Association for the Assessment and Accreditation of Laboratory Animal multiple processes. The potential compensatory interplay between Care (AAALAC) and the recommendations of Federation of European desmin and αΒ-crystallin is further supported by the fact that both of Laboratory Animal Science Association (FELASA). them associate with important mitochondrial proteins. Our data indicate that desmin plays a possible role in the formation and/or Southern and western blotting For the identification of transgenes by southern blotting, a fragment of stabilization of Mic60 macromolecular complexes. In the absence of α 32 α MHC was isolated and used to generate P-labeled probes by random desmin, this role is restored by overexpressed B-crystallin. As priming. discussed above, Mic60 is crucial for the formation and function of For western blotting analysis, proteins were transferred to polyvinylidene the extended cristae membrane structures, the main sites of ATP fluoride (PVDF) membrane (Whatman) and probed with the following synthesis by oxidative phosphorylation (Gilkerson et al., 2003; antibodies: anti-αB-Crystallin (Stressgen, SPA223, 1:2000; Santa Cruz,

Vogel et al., 2006; Wurm and Jakobs, 2006). In addition, it is sc-22744, 1:1000), anti-desmin (Abcam, ab8592, 1:1000), anti-GAPDH Journal of Cell Science

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(Ambion, AM4300, 1:4000), anti-ANT (Santa Cruz, sc-9299, 1:200), equipped with a HC-PL-APO-CS 63× oil objective and LAS-AF anti-ATP5B (Mitosciences, ab5432, 1:500), anti-mitofilin (Novus acquisition software (Leica). Biologicals, NB100-1919, 1:1000; Abcam, ab110329, 1:1000), anti- NDUFS2 (Thermo Scientific, PA5-19342, 1:500), anti-SDHA (ab14715, Swimming trial 1:1000), anti-COXIV (ab33985, 1:1000), anti-VDAC1 (ab14734, 1:1000; Mice were exercised twice a day using a swimming protocol, as previously ab15895, 1:1000; both Abcam), anti-mitofusin-2 (Sigma, M6444, 1:1000) described (Milner et al., 1999). The swimming program began at 10 min/day and anti-MnSOD (BD Biosciences, M9920, 1:500) antibodies. Secondary and was increased by 10 min daily to a maximum of 1 h. The twice-a-day horseradish peroxidase (HRP)-conjugated antibodies were purchased from 1 h exercise was continued for 9 days, and subsequently was increased to Sigma-Aldrich. Proteins were visualized using chemiluminescence (ECL 90 min per session. The twice-daily 90 min exercise was continued for kit, Amersham). Films were scanned using a GS800 densitometer (Bio-Rad) 9 days. In the case of αB-crystallin localization studies, a three-day protocol, and quantified using Quantity one software (Bio-Rad). during which the mice were exercised twice for 30 min the first day and twice daily for 1 h the next two days. Histology For the evaluation of fibrosis, Masson’s-trichrome-stained paraffin sections Echocardiography were photographed and subjected to ImageJ software analysis. The results Mice were anesthetized through intraperitoneal injection of ketamine were expressed as the percentage of fibrosis in a given tissue section. For the (100 mg/kg of bodyweight). Echocardiographic studies were performed evaluation of inflammation, hearts were observed under a Stemi 2000-C using a Vivid 7 GE ultrasound system with a 13-MHz linear transducer. stereomicroscope (Zeiss) and were graded 0–4, according to the presence of Two-dimensional targeted M-mode imaging was obtained from the short- inflammatory infiltrate, as previously described (Psarras et al., 2012) – 0=no axis view at the level of greatest left ventricular dimension. Images were inflammatory infiltrate; 1=one inflammatory infiltrate; 2=more than one analyzed using the Echopac PC SW 3.1.3 software (GE Healthcare). Data inflammatory infiltrates appearing in limited areas of the myocardial wall; were expressed as mean±s.d. for continuous variables and analyzed by using 3=multiple inflammatory infiltrates extended in multiple areas; 4=multiple Statview 5.0 (Abacus Concepts). Statistical comparisons were performed extended inflammatory infiltrates eventually covering both the anterior and using ANOVA with Bonferroni or Dunn post-hoc test, or the unpaired posterior wall. Student’s t-test where appropriate.

Evans Blue staining Adult cardiomyocyte isolation The mice were injected intraperitoneally with EBD (250 μg/g of body Isolation was performed using a modified protocol (Diokmetzidou et al., weight in 1×PBS). After 24 h, the heart was embedded in optimum-cutting 2016) based on standard procedures (O’Connell et al., 2007). temperature (OCT) compound (VWR) and snap frozen in liquid nitrogen. EBD staining was evaluated by using the ImageJ software, and the results Measurement of mitochondrial membrane potential, mPTP were expressed as the percentage of EBD-stained area in the image. opening and ROS Adult cardiomyocytes were incubated with 20 nM TMRM (Invitrogen) for Electron microscopy 15 min at 37°C, washed and examined by using time-lapse confocal Mice were treated with heparin for 30 min and euthanized, and the heart was scanning for 20 min with a 1-min interval (TCS SP5, Leica) by excitation at perfused with 2.5% glutaraldehyde in 0.1 M phosphate puffer, pH 7.4, fixed 568 nm and emission at 580–640 nm. 5 μM cyclosporin A (Invitrogen) was overnight and post-fixed with 1% osmium tetroxide for 1 h at 4°C, added with TMRM to confirm mPTP opening. For the ROS measurement, – dehydrated, embedded in epon araldite resin mixture and allowed to the cardiomyocytes were incubated with 20 μΜ CM-H2DCFDA polymerize at 60°C for 24 h. Ultrathin sections (65–70 nm) (Leica EM-UC7 (Invitrogen) for 30 min at 37°C, washed and examined using a 488-nm ultramicrotome, Leica) were examined with a Philips 201C transmission argon-ion laser excitation and emission at 503–555 nm. A HCX-PL-APO- electron microscope and photographed on Agfa Copex HDP13 microfilm. CS-20 ×0.70 dry UV objective and LAS-AF acquisition software (Leica) For immunogold labeling, mice were perfused with cold 4% were used in both cases. The acquired images were analyzed using the paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer, pH Volocity 5 software (Perkin Elmer). 7.4. Hearts were post fixed in the same fixative for 2 h and rinsed in 0.1 M Ca2+-induced swelling of mitochondria was performed as previously phosphate buffer. The dehydration and embedding of tissue samples was described (Weisleder et al., 2004). 5 μΜ CsA was added to mitochondria performed using the progressive lowering of temperature (PLT) method using 5 min before CaCl2. a Leica EM AFS (Leica), as described previously (Robertson et al., 1992). Tissue fractionation, mitochondrial purification and isolation of Double immunogold labeling MAMs Mounted ultrathin sections were subjected to the post-embedding Fractionation of mouse or rat hearts and the isolation of MAMs and pure immunogold procedure, as previously described (Havaki et al., 2003; mitochondria were performed by differential centrifugation at 4°C, as Diokmetzidou et al., 2016). All incubations were performed using the previously described (Wieckowski et al., 2009; Diokmetzidou et al., 2016; automated immunogold labeling Leica EM IGL instrument. Primary Frezza et al., 2007). antibodies (VDAC, Abcam, ab147234, 1:10; desmin, Abcam, ab8592, 1:20) were incubated overnight at 4°C, and samples were incubated for 1 h at GSH measurements room temperature with 10-nm-gold-conjugated secondary antibodies The GSH concentration was measured in hearts of 6-month-old mice, as against polyclonal IgG particles (1:40) or 25-nm-gold-conjugated previously described (Rahman et al., 2006). The hearts were weighed, secondary antibodies against monoclonal IgG (1:40). Sections were homogenized in 5% sulfosalicilic acid and centrifuged at 10,000 g for examined with a Philips 420 transmission electron microscope at 60 kV 10 min at 4°C. The supernatant was removed and used for the GSH assay and photographed with a Megaview G2 CCD camera (Olympus SIS). based on that of Griffith (1980).

Immunofluorescence Determination of ATP levels Frozen mouse cardiac sections were used for immunolabeling, as ATP content was measured using the Aposensor ATP assay kit (Biovision) previously described (Diokmetzidou et al., 2016). Primary antibodies in a luminometer (Berthold) according to the manufacturer’s instructions. against αB-crystallin (Stressgen, SPA223, 1:100; Santa Cruz, sc-22744, 1:50) and α-actinin (Sigma-Aldrich, Α7811, 1:1000) were incubated Two-dimensional native blue and SDS PAGE overnight at 4°C, and with secondary antibodies (conjugated to Alexa- Mouse cardiac mitochondria were solubilized in native solubilization buffer Fluor-594 or Alexa-Fluor-488, Invitrogen) for 1 h at room temperature. pH 7 (20 mM Bis-Tris, 500 mM aminocaproic acid, 20 mM NaCl, 2 mM

The sections were examined using a Leica TCS SP5 confocal microscope EDTA pH 8, 10% glycerol, 2 mM phenylmethylsulfonyl fluoride, protease Journal of Cell Science

3717 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 3705-3720 doi:10.1242/jcs.192203 inhibitor cocktail) with 4 g digitonin per g of protein for 30 min on ice and pH 8, 10% glycerol, 2 mM PMSF, protease inhibitor cocktail), containing analyzed in a 3–12% native gel (Serva). Individual lanes were excised, 4 g digitonin/g of protein, for 30 min on ice and were pre-cleared for 3 h at incubated in equilibration buffer (125 mM Tris-HCl, pH 6.8, and 1% SDS) 4°C and used as previously described (Diokmetzidou et al., 2016). The for 20 min and analyzed in the second dimension by 10% gel SDS-PAGE. bound proteins were eluted by heating at 97°C for 10 min in 2× Proteins were transferred to PVDF membrane (Whatman) and probed with electrophoresis loading buffer or, in the case of GST pull down, with the desired antibodies. 20 mM reduced GSH in 50 mM Tris-HCl, pH 8, for 30 min at room temperature. The eluates were analyzed by western blotting. Two-dimensional gel electrophoresis Two-dimensional gel electrophoresis was performed as previously Protease accessibility assay described (Makridakis et al., 2010) with the following specifications. One hundred μg (1 mg/ml) of crude mouse cardiac mitochondria were Mitochondrial fractions were solubilized in the isoelectric focusing (IEF) suspended in MSE buffer (225 mM mannitol, 75 mM sucrose, 1 mm EGTA, sample buffer [7 M urea, 2 M thiourea, 4% CHAPS, 1% dithioerythreitol, 1 mM Tris-HCl pH 7.4) or osmotic buffer (20 mM KCl) with or without 0.5% 2% ampholytes (pH range: 3–10)] and resolved (100 μg per sample) on Triton X-100 and incubated with 2 μg/ml proteinase K for 1 h on ice. The 7-cm strips, pH range 3–10 non-linear (Bio-Rad), using the in gel reaction was stopped with 4 mM (final) PMSF. Samples were loaded with rehydration method. IEF was conducted for about 12,500 volt-hours. 2× SDS-PAGE loading buffer and analyzed by western blotting. Strips were then incubated in equilibration buffer (6 M urea, 50 mM Tris- HCl pH 8.8, 30% glycerol, 2% SDS) containing 0.5% dithioerythreitol for Statistical analysis 45 min with gentle agitation, followed by a second incubation with All data are expressed as mean±s.e.m. unless otherwise stated. Statistical equilibration buffer containing 4.32% iodoacetamide. Second dimensional comparisons were performed using ANOVA with Bonferroni or Dunn post- analysis was performed on a 12% SDS-PAGE gel, and the gels were stained hoc test, or unpaired Student’s t-test where appropriate. A P-value<0.05 was with Coomassie Colloidal Blue stain (Candiano et al., 2004). considered significant. Graphics and statistical analysis were performed using Sigma Plot 10.0 (Systat Software). Quantification of spots Gels were scanned on a GS-800 imaging densitometer (Bio-Rad) in Acknowledgements transmission mode, and image analysis was performed with the PD Quest 8 We thank very much S. Pagkakis and E. Rigana for their help with imaging. We are software package (Bio-Rad). Individual protein spot quantification was grateful to E. Mavroidis and I. Kostavasili for constant assistance throughout this normalized based on the total intensity of the spots in the gel and expressed work. We also thank Prof. William James Craigen and N. Flytzanis for valuable in ppm. Statistical analysis was conducted using the Mann–Whitney and comments on the manuscript. Student’s t-test. Competing interests The authors declare no competing or financial interests. Protein identification with MALDI-TOF MS Protein spots were excised manually or automatically with the use of the ProteineerSp Protein picker (BrukerDaltonics), destained (30% acetonitrile, Author contributions A.D. designed and performed experiments, analyzed data and wrote the 50 mM NH HCO ), washed, dried and trypsinized overnight (trypsin, 4 3 manuscript; E.S. and M.T. performed experiments; I.K. performed the electron proteomics-grade, Roche). Peptides were extracted (50% acetonitrile, microscopy experiments; M.M. and A. Vlahou performed and analyzed the 0.1% trifluoroacetic acid) and applied onto the matrix-assisted laser proteomic experiments; S.G. helped with transgenic mice generation; A. Varela and desorption ionization (MALDI) target using the dry droplet method. C.H.D. performed and analyzed the echocardiography; Y.C. directed the research Peptide masses were determined by using MALDI time-of-flight mass project, analyzed the data and wrote the manuscript. I.K. and M.T. made equal spectrometry (MALDI-TOF)–TOF-MS (Ultraflex TOF/TOF instrument, contributions to the work. BrukerDaltonics). The peak list was created with Flexanalysis v3.3 software (Bruker); smoothing was applied with the Savitzky–Golay algorithm (width Funding 0.2 m/z, cycle number 1), and a signal:noise threshold ratio of 2.5 was This work was supported by ESPA 2007-2013 grants from the Greek General allowed. For peptide matching (Mascot Server 2; Matrix Science), the Secretariat of Research and Development – Ministerium of Education [grant following settings were used: monoisotopic mass, one miscleavage site, numbers PENED 01ED371, EPAN YB-22, PEP ATT-39 and ESPA 09SYN-21-965 carbamidomethylation of cysteine as fixed and oxidation of methionine as to Y.C.], including a grant of Excellence II/ARISTEIA II 5342 (to Y.C.); a grant from variable modifications. Stringent criteria were used for protein identification the US National Institutes of Health [grant number RO1 AR39617 to Y.C.]; and in part by a European Commission grant [grant number FP7/REGPOT-2008-1 with a maximum allowed mass error of 25 ppm and a minimum of four (Transmed)] to A.V. Deposited in PMC for release after 12 months. matching peptides. False identity probability was usually lower than 10−5. Data analyzed with the same settings as described above, using a sequence Supplementary information scrambled version of Swiss-Prot data base generated by the decoy-generating Supplementary information available online at script that is available at Matrix Science, provided no identifications. http://jcs.biologists.org/lookup/doi/10.1242/jcs.192203.supplemental

Expression and purification of GST-tagged proteins – – – References Desmin mutants head, amino acids 1 108 (1 324 bp); head-rod, amino Arai, H. and Atomi, Y. (1997). Chaperone activity of alpha B-crystallin – – – – acids 1 412 (1 1236 bp); rod, amino acids 109 412 (324 1236 bp); rod- suppresses tubulin aggregation through complex formation. Cell Struct. Funct. tail, amino acids 109–470 (324–1410 bp); tail, amino acids 413–470 (1237– 22, 539-544. 1410 bp). VDAC open reading frame and mutants – ΔNterm, amino acids Bennardini, F., Wrzosek, A. and Chiesi, M. (1992). Alpha B-crystallin in 33–296 (99–888 bp) lacking the N-terminal α-helix; Δβ1–β4, lacks amino cardiac tissue. Association with actin and desmin filaments. Circ. Res. 71, acids 34–81 (102–243 bp), thus lacking β-barrels 1–4. Sequences were 288-294. Bhuiyan, M. S., Pattison, J. S., Osinska, H., James, J., Gulick, J., Mclendon, isolated by using PCR with wild-type mouse heart cDNA as template and P. M., Hill, J. A., Sadoshima, J. and Robbins, J. (2013). Enhanced autophagy DNA Q5 high fidelity polymerase and sub-cloned into pGEX-4T-3. The ameliorates cardiac proteinopathy. J. Clin. Invest. 123, 5284-5297. recombinant polypeptides were expressed in BL21 bacteria, and the proteins Cabet, E., Batonnet-Pichon, S., Delort, F., Gausseres,̀ B., Vicart, P. and were isolated as previously described (Kouloumenta et al., 2007; Lilienbaum, A. (2015). Antioxidant treatment and induction of autophagy Diokmetzidou et al., 2016) cooperate to reduce desmin aggregation in a cellular model of desminopathy. PLoS ONE 10, e0137009. Candiano, G., Bruschi, M., Musante, L., Santucci, L., Ghiggeri, G. M., GST pull down and co-immunoprecipitation Carnemolla, B., Orecchia, P., Zardi, L. and Righetti, P. G. (2004). Blue silver: Mouse cardiac mitochondria (5 mg) were solubilized in native buffer pH 7 a very sensitive colloidal Coomassie G-250 staining for proteome analysis.

(20 mM Bis-Tris, 500 mM aminocaproic acid, 20 mM NaCl, 2 mM EDTA, Electrophoresis 25, 1327-1333. Journal of Cell Science

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Capetanaki, Y., Bloch, R. J., Kouloumenta, A., Mavroidis, M. and Psarras, S. Kouloumenta, A., Mavroidis, M. and Capetanaki, Y. (2007). Proper perinuclear (2007). Muscle intermediate filaments and their links to membranes and localization of the TRIM-like protein myospryn requires its binding partner desmin. membranous organelles. Exp. Cell Res. 313, 2063-2076. J. Biol. Chem. 282, 35211-35221. Capetanaki, Y., Papathanasiou, S., Diokmetzidou, A., Vatsellas, G. and Tsikitis, Li, Z., Colucci-Guyon, E., Pinçon-Raymond, M., Mericskay, M., Pournin, S., M. (2015). Desmin related disease: a matter of cell survival failure. Curr. Opin. Cell Paulin, D. and Babinet, C. (1996). Cardiovascular lesions and skeletal myopathy Biol. 32, 113-120. in mice lacking desmin. Dev. Biol. 175, 362-366. Cogliati, S., Frezza, C., Soriano, M. E., Varanita, T., Quintana-Cabrera, R., Li, J., Horak, K. M., Su, H., Sanbe, A., Robbins, J. and Wang, X. (2011). Corrado, M., Cipolat, S., Costa, V., Casarin, A., Gomes, L. C. et al. (2013). Enhancement of proteasomal function protects against cardiac proteinopathy and Mitochondrial cristae shape determines respiratory chain supercomplexes ischemia/reperfusion injury in mice. J. Clin. Invest. 121, 3689-3700. assembly and respiratory efficiency. Cell 155, 160-171. Makridakis, M., Roubelakis, M. G., Bitsika, V., Dimuccio, V., Samiotaki, M., Darshi, M., Mendiola, V. L., Mackey, M. R., Murphy, A. N., Koller, A., Perkins, Kossida, S., Panayotou, G., Coleman, J., Candiano, G., Anagnou, N. P. et al. G. A., Ellisman, M. H. and Taylor, S. S. (2011). ChChd3, an inner mitochondrial (2010). Analysis of secreted proteins for the study of bladder cancer cell membrane protein, is essential for maintaining crista integrity and mitochondrial aggressiveness. J. Proteome Res. 9, 3243-3259. function. J. Biol. Chem. 286, 2918-2932. Maloyan, A., Sanbe, A., Osinska, H., Westfall, M., Robinson, D., Imahashi, K.-i., Diokmetzidou, A., Tsikitis, M., Nikouli, S., Kloukina, I., Tsoupri, E., Murphy, E. and Robbins, J. (2005). Mitochondrial dysfunction and apoptosis α Papathanasiou, S., Psarras, S., Mavroidis, M. and Capetanaki, Y. (2016). underlie the pathogenic process in -B-crystallin desmin-related cardiomyopathy. Circulation 112, 3451-3461. Strategies to study Desmin in cardiac muscle and culture systems. Methods Martindale, J. J., Wall, J. A., Martinez-Longoria, D. M., Aryal, P., Rockman, H. A., Enzymol. 568, 427-459. Guo, Y., Bolli, R. and Glembotski, C. C. (2005). Overexpression of mitogen- Dudkina, N. V., Sunderhaus, S., Braun, H.-P. and Boekema, E. J. (2006). activated protein kinase kinase 6 in the heart improves functional recovery from Characterization of dimeric ATP synthase and cristae membrane ultrastructure ischemia in vitro and protects against myocardial infarction in vivo. J. Biol. Chem. from Saccharomyces and Polytomella mitochondria. FEBS Lett. 580, 3427-3432. 280, 669-676. Fountoulakis, M., Soumaka, E., Rapti, K., Mavroidis, M., Tsangaris, G., Maris, Mavroidis, M. and Capetanaki, Y. (2002). Extensive induction of important A., Weisleder, N. and Capetanaki, Y. (2005). Alterations in the heart mediators of fibrosis and dystrophic calcification in desmin-deficient mitochondrial proteome in a desmin null heart failure model. J. Mol. Cell. cardiomyopathy. Am. J. Pathol. 160, 943-952. Cardiol. 38, 461-474. Mavroidis, M., Davos, C. H., Psarras, S., Varela, A., Athanasiadis, N. C., Frezza, C., Cipolat, S., Martins De Brito, O., Micaroni, M., Beznoussenko, G. V., Katsimpoulas, M., Kostavasili, I., Maasch, C., Vater, A., Van Tintelen, J. P. Rudka, T., Bartoli, D., Polishuck, R. S., Danial, N. N., De Strooper, B. et al. et al. (2015). Complement system modulation as a target for treatment of (2006). OPA1 controls apoptotic cristae remodeling independently from arrhythmogenic cardiomyopathy. Basic Res. Cardiol. 110, 27. mitochondrial fusion. Cell 126, 177-189. McLendon, P. M. and Robbins, J. (2011). Desmin-related cardiomyopathy: an Frezza, C., Cipolat, S. and Scorrano, L. (2007). Organelle isolation: functional unfolding story. Am. J. Physiol. Heart Circ. Physiol. 301, H1220-H1228. mitochondria from mouse liver, muscle and cultured fibroblasts. Nat. Protoc. 2, Milner, D. J., Weitzer, G., Tran, D., Bradley, A. and Capetanaki, Y. (1996). 287-295. Disruption of muscle architecture and myocardial degeneration in mice lacking Gieffers, C., Korioth, F., Heimann, P., Ungermann, C. and Frey, J. (1997). desmin. J. Cell Biol. 134, 1255-1270. Mitofilin is a transmembrane protein of the inner mitochondrial membrane Milner, D. J., Taffet, G. E., Wang, X., Pham, T., Tamura, T., Hartley, C., Gerdes, expressed as two isoforms. Exp. Cell Res. 232, 395-399. A. M. and Capetanaki, Y. (1999). The absence of desmin leads to cardiomyocyte Gilkerson, R. W., Selker, J. M. L. and Capaldi, R. A. (2003). The cristal membrane hypertrophy and cardiac dilation with compromised systolic function. J. Mol. Cell. of mitochondria is the principal site of oxidative phosphorylation. FEBS Lett. 546, Cardiol. 31, 2063-2076. 355-358. Milner, D. J., Mavroidis, M., Weisleder, N. and Capetanaki, Y. (2000). Desmin Giorgio, V., Von Stockum, S., Antoniel, M., Fabbro, A., Fogolari, F., Forte, M., cytoskeleton linked to muscle mitochondrial distribution and respiratory function. Glick, G. D., Petronilli, V., Zoratti, M., Szabo, I. et al. (2013). Dimers of J. Cell Biol. 150, 1283-1298. mitochondrial ATP synthase form the permeability transition pore. Proc. Natl. Minauro-Sanmiguel, F., Wilkens, S. and Garcia, J. J. (2005). Structure of dimeric Acad. Sci. USA 110, 5887-5892. mitochondrial ATP synthase: novel F0 bridging features and the structural basis of Goldfarb, L. G., Park, K.-Y., Cervenaková, L., Gorokhova, S., Lee, H.-S., mitochondrial cristae biogenesis. Proc. Natl. Acad. Sci. USA 102, 12356-12358. Vasconcelos, O., Nagle, J. W., Semino-Mora, C., Sivakumar, K. and Dalakas, Mitra, A., Basak, T., Datta, K., Naskar, S., Sengupta, S. and Sarkar, S. (2013). M. C. (1998). Missense mutations in desmin associated with familial cardiac and Role of alpha-crystallin B as a regulatory switch in modulating cardiomyocyte skeletal myopathy. Nat. Genet. 19, 402-403. apoptosis by mitochondria or endoplasmic reticulum during cardiac hypertrophy Golenhofen, N., Ness, W., Koob, R., Htun, P., Schaper, W. and Drenckhahn, D. and myocardial infarction. Cell Death Dis. 4, e582. (1998). Ischemia-induced phosphorylation and translocation of stress protein Naon, D. and Scorrano, L. (2014). At the right distance: ER-mitochondria alpha B-crystallin to Z lines of myocardium. Am. J. Physiol. 274, H1457-H1464. juxtaposition in cell life and death. Biochim. Biophys. Acta 1843, 2184-2194. Griffith, O. W. (1980). Determination of glutathione and glutathione disulfide using Narula, N., Zaragoza, M. V., Sengupta, P. P., Li, P., Haider, N., Verjans, J., glutathione reductase and 2-vinylpyridine. Anal. Biochem. 106, 207-212. Waymire, K., Vannan, M. and Wallace, D. C. (2011). Adenine nucleotide Gupta, M. K., Gulick, J., Liu, R., Wang, X., Molkentin, J. D. and Robbins, J. translocase 1 deficiency results in dilated cardiomyopathy with defects in (2014). Sumo E2 enzyme UBC9 is required for efficient protein quality control in myocardial mechanics, histopathological alterations, and activation of cardiomyocytes. Circ. Res. 115, 721-729. apoptosis. JACC Cardiovasc Imaging 4, 1-10. Harner, M., Körner, C., Walther, D., Mokranjac, D., Kaesmacher, J., Welsch, U., Nicholl, I. D. and Quinlan, R. A. (1994). Chaperone activity of alpha-crystallins Griffith, J., Mann, M., Reggiori, F. and Neupert, W. (2011). The mitochondrial modulates intermediate filament assembly. EMBO J. 13, 945-953. O’connell, T. D., Rodrigo, M. C. and Simpson, P. C. (2007). Isolation and culture of contact site complex, a determinant of mitochondrial architecture. EMBO J. 30, adult mouse cardiac myocytes. Methods Mol. Biol. 357, 271-296. 4356-4370. Odgren, P. R., Toukatly, G., Bangs, P. L., Gilmore, R. and Fey, E. G. (1996). Havaki, S., Kittas, C., Marinos, E., Dafni, U., Sotiropoulou, C., Goutas, N., Molecular characterization of mitofilin (HMP), a mitochondria-associated protein Voloudakis-Baltatzis, I., Vassilaros, S. D., Athanasiou, E. and Arvanitis, D. L. with predicted coiled coil and intermembrane space targeting domains. J. Cell Sci. (2003). Ultrastructural immunostaining of infiltrating ductal breast carcinomas with 109, 2253-2264. the monoclonal antibody H: a comparative study with cytokeratin 8. Ultrastruct. Panagopoulou, P., Davos, C. H., Milner, D. J., Varela, E., Cameron, J., Mann, Pathol. 27, 393-407. D. L. and Capetanaki, Y. (2008). Desmin mediates TNF-alpha-induced ̀ Hnia, K., Tronchere, H., Tomczak, K. K., Amoasii, L., Schultz, P., Beggs, A. H., aggregate formation and intercalated disk reorganization in heart failure. J. Cell Payrastre, B., Mandel, J. L. and Laporte, J. (2011). Myotubularin controls Biol. 181, 761-775. desmin intermediate filament architecture and mitochondrial dynamics in human Papathanasiou, S., Rickelt, S., Soriano, M. E., Schips, T. G., Maier, H. J., Davos, and mouse skeletal muscle. J. Clin. Invest. 121, 70-85. C. H., Varela, A., Kaklamanis, L., Mann, D. L. and Capetanaki, Y. (2015). Tumor Hoppins, S., Collins, S. R., Cassidy-Stone, A., Hummel, E., Devay, R. M., necrosis factor-α confers cardioprotection through ectopic expression of keratins Lackner, L. L., Westermann, B., Schuldiner, M., Weissman, J. S. and Nunnari, K8 and K18. Nat. Med. 21, 1076-1084. J. (2011). A mitochondrial-focused genetic interaction map reveals a scaffold-like Pattison, J. S., Osinska, H. and Robbins, J. (2011). Atg7 induces basal autophagy complex required for inner membrane organization in mitochondria. J. Cell Biol. and rescues autophagic deficiency in CryABR120G cardiomyocytes. Circ. Res. 195, 323-340. 109, 151-160. Houck, S. A. and Clark, J. I. (2010). Dynamic subunit exchange and the regulation Paumard, P., Vaillier, J., Coulary, B., Schaeffer, J., Soubannier, V., Mueller, of microtubule assembly by the stress response protein human αB crystallin. D. M., Brethes,̀ D., Di Rago, J.-P. and Velours, J. (2002). The ATP synthase is PLoS ONE 5, e11795. involved in generating mitochondrial cristae morphology. EMBO J. 21, 221-230. Iwaki, T., Kume-Iwaki, A., Liem, R. K. H. and Goldman, J. E. (1989). αB-crystallin Perng, M. D., Cairns, L., Van Den, I. P., Prescott, A., Hutcheson, A. M. and is expressed in non-lenticular tissues and accumulates in Alexander’s disease Quinlan, R. A. (1999). Intermediate filament interactions can be altered by HSP27

brain. Cell 57, 71-78. and alphaB-crystallin. J. Cell Sci. 112, 2099-2112. Journal of Cell Science

3719 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 3705-3720 doi:10.1242/jcs.192203

Psarras, S., Mavroidis, M., Sanoudou, D., Davos, C. H., Xanthou, G., Varela, Vogel, F., Bornhövd, C., Neupert, W. and Reichert, A. S. (2006). Dynamic A. E., Panoutsakopoulou, V. and Capetanaki, Y. (2012). Regulation of adverse subcompartmentalization of the mitochondrial inner membrane. J. Cell Biol. 175, remodelling by osteopontin in a genetic heart failure model. Eur. Heart J. 33, 237-247. 1954-1963. Vogelpohl, I., Vetter, R., Heger, J., Ebermann, L., Euler, G., Schultheiss, H. P. Rahman, I., Kode, A. and Biswas, S. K. (2006). Assay for quantitative and Dörner, A. (2011). Transgenic overexpression of heart-specific adenine determination of glutathione and glutathione disulfide levels using enzymatic nucleotide translocase 1 positively affects contractile function in cardiomyocytes. recycling method. Nat. Protoc. 1, 3159-3165. Cell. Physiol. Biochem. 27, 121-128. Rajasekaran, N. S., Connell, P., Christians, E. S., Yan, L. J., Taylor, R. P., Orosz, Von der Malsburg, K., Müller, J. M., Bohnert, M., Oeljeklaus, S., Kwiatkowska, A., Zhang, X. Q., Stevenson, T. J., Peshock, R. M., Leopold, J. A. et al. (2007). P., Becker, T., Loniewska-Lwowska, A., Wiese, S., Rao, S., Milenkovic, D. Human alpha B-crystallin mutation causes oxido-reductive stress and protein et al. (2011). Dual role of mitofilin in mitochondrial membrane organization and aggregation cardiomyopathy in mice. Cell 130, 427-439. protein biogenesis. Dev. Cell 21, 694-707. Reipert, S., Steinböck, F., Fischer, I., Bittner, R. E., Zeöld, A. and Wiche, G. Wang, X. and Robbins, J. (2006). Heart failure and protein quality control. Circ. (1999). Association of mitochondria with plectin and desmin intermediate Res. 99, 1315-1328. filaments in striated muscle. Exp. Cell Res. 252, 479-491. Wang, X., Osinska, H., Klevitsky, R., Gerdes, A. M., Nieman, M., Lorenz, J., Robertson, D., Monaghan, P., Clarke, C. and Atherton, A. J. (1992). An appraisal Hewett, T. and Robbins, J. (2001). Expression of R120G-alphaB-crystallin of low-temperature embedding by progressive lowering of temperature into causes aberrant desmin and alphaB-crystallin aggregation and cardiomyopathy in Lowicryl HM20 for immunocytochemical studies. J. Microsc. 168, 85-100. mice. Circ. Res. 89, 84-91. Singh, B. N., Rao, K. S., Ramakrishna, T., Rangaraj, N. and Rao Ch, M. (2007). Wang, X., Klevitsky, R., Huang, W., Glasford, J., Li, F. and Robbins, J. (2003). Association of αB-crystallin, a small heat shock protein, with actin: role in AlphaB-crystallin modulates protein aggregation of abnormal desmin. Circ. Res. modulating actin filament dynamics in vivo. J. Mol. Biol. 366, 756-767. 93, 998-1005. Strauss, M., Hofhaus, G., Schröder, R. R. and Kühlbrandt, W. (2008). Dimer Weisleder, N., Taffet, G. E. and Capetanaki, Y. (2004). Bcl-2 overexpression ribbons of ATP synthase shape the inner mitochondrial membrane. EMBO J. 27, corrects mitochondrial defects and ameliorates inherited desmin null 1154-1160. cardiomyopathy. Proc. Natl. Acad. Sci. USA 101, 769-774. Stromer, M. H. and Bendayan, M. (1990). Immunocytochemical identification of Wettstein, G., Bellaye, P. S., Micheau, O. and Bonniaud, P. (2012). Small heat cytoskeletal linkages to smooth muscle cell nuclei and mitochondria. Cell Motil. shock proteins and the cytoskeleton: an essential interplay for cell integrity? Cytoskeleton 17, 11-18. Int. J. Biochem. Cell Biol. 44, 1680-1686. Su, H., Li, J., Zhang, H., Ma, W., Wei, N., Liu, J. and Wang, X. (2015). COP9 Wieckowski, M. R., Giorgi, C., Lebiedzinska, M., Duszynski, J. and Pinton, P. signalosome controls the degradation of cytosolic misfolded proteins and protects (2009). Isolation of mitochondria-associated membranes and mitochondria from against cardiac proteotoxicity. Circ. Res. 117, 956-966. animal tissues and cells. Nat. Protoc. 4, 1582-1590. Subramaniam, A., Jones, W. K., Gulick, J., Wert, S., Neumann, J. and Robbins, Winter, L., Abrahamsberg, C. and Wiche, G. (2008). Plectin isoform 1b mediates J. (1991). Tissue-specific regulation of the alpha-myosin heavy chain gene mitochondrion-intermediate filament network linkage and controls organelle promoter in transgenic mice. J. Biol. Chem. 266, 24613-24620. shape. J. Cell Biol. 181, 903-911. Van Der Laan, M., Bohnert, M., Wiedemann, N. and Pfanner, N. (2012). Role of Wisniewski, T. and Goldman, J. E. (1998). Alpha B-crystallin is associated with MINOS in mitochondrial membrane architecture and biogenesis. Trends Cell Biol. intermediate filaments in astrocytoma cells. Neurochem. Res. 23, 385-392. 22, 185-192. Wurm, C. A. and Jakobs, S. (2006). Differential protein distributions define two sub- Van Spaendonck-Zwarts, K. Y., Van Hessem, L., Jongbloed, J. D. H., De Walle, compartments of the mitochondrial inner membrane in yeast. FEBS Lett. 580, H. E. K., Capetanaki, Y., Van Der Kooi, A. J., Van Langen, I. M., Van Den Berg, 5628-5634. M. P. and Van Tintelen, J. P. (2011). Desmin-related myopathy. Clin. Genet. 80, Xie, J., Marusich, M. F., Souda, P., Whitelegge, J. and Capaldi, R. A. (2007). The 354-366. mitochondrial inner membrane protein mitofilin exists as a complex with SAM50, Vicart, P., Caron, A., Guicheney, P., Li, Z., Prévost, M.-C., Faure, A., Chateau, D., metaxins 1 and 2, coiled-coil-helix coiled-coil-helix domain-containing protein 3 Chapon, F., Tomé, F., Dupret, J.-M. et al. (1998). A missense mutation in the and 6 and DnaJC11. FEBS Lett. 581, 3545-3549. alphaB-crystallin chaperone gene causes a desmin-related myopathy. Nat. Zick, M., Rabl, R. and Reichert, A. S. (2009). Cristae formation-linking Genet. 20, 92-95. ultrastructure and function of mitochondria. Biochim. Biophys. Acta 1793, 5-19. Journal of Cell Science

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