© 2016. Published by The Company of Biologists Ltd.

Desmin and αB-Crystallin Interplay in 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,&

1 Center of Basic Research, 2Center of Systems Biology, 3Center of Clinical, Experimental Surgery &

Translational Research, Biomedical Research Foundation, Academy of Athens, Athens 11527, Greece

# Contributed equally

& Corresponding author:

Yassemi Capetanaki

Center of Basic Research

Biomedical Research Foundation

Academy of Athens

11527 Athens, Greece

Tel: +30-210-6597212

Fax: +30-210-6597545

e-mail: ycapetanaki@bioacademy.gr

Key words: intermediate filaments, small heat shock protein, cytoskeleton, heart failure,

mitochondria, cristae

Summary Statement

We found that both desmin and its partner chaperon αB-crystallin associate to mitochondria-

sarcoplasmic reticulum contact sites and stabilize MICOS supercomplexes, thus contributing to

proper mitochondrial cristae structure-function and cardiomyocyte survival.

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JCS Advance Online Article. Posted on 26 August 2016

Abstract

The association of desmin with αΒ-crystallin, and the fact that mutations in either one of them lead to

heart failure in humans and mice, suggests a potential compensatory interplay between them in

cardioprotection. To address this hypothesis, we investigated the consequences of αΒ-crystallin

overexpression in the desmin deficient (des-/-) mouse model, which possesses a combination of the

pathologies found in most cardiomyopathies, with mitochondrial defects as a hallmark. We

demonstrated that cardiac specific αΒ-crystallin overexpression ameliorates all these defects and

significantly improves cardiac function to almost wild type levels. Protection by αΒ-crystallin

overexpression is linked to maintenance of proper mitochondrial protein levels, inhibition of abnormal

mitochondrial permeability transition pore activation and maintenance of mitochondrial membrane

potential (Δψ). Furthermore, we found that both desmin and αΒ-crystallin are localized at SR-

mitochondria associated membranes, where they interact with VDAC, Mic60, the core component of

MICOS (mitochondrial contact site and cristae organizing system) complex and ATP synthase,

suggesting that these associations could be crucial in mitoprotection at different levels.

List of Symbols and Abbreviations

ANT: adenine nucleotide translocase

CM-H2DCFDA: 5-(and-6)-chloromethyl-2′,7′-dichlorohydrofluorescein diacetate, acetyl ester

COXIV: cytochrome C oxidase IV

CsA: cyclosporin A

DCM: dilated cardiomyopathy

des-/-: desmin knockout mice

des-/-αΒCry: desmin knockout mice overexpressing αΒ-Crystallin

GAPDH: glyceraldehyde 3-phosphate dehydrogenase

ID: intercalated disc

IF: intermediate filament

IMM: inner mitochondrial membrane

LM: light membranes

MAMs: mitochondria-associated sarcoplasmic reticulum membranes

MICOS: mitochondrial contact site and cristae organizing system

mPTP: mitochondrial permeability transition pore

OMM: outer mitochondrial membrane

ROS: reactive oxygen species

SR: sarcoplasmic reticulum

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TMRM: tetramethylrhodamine methyl ester

VDAC: voltage dependent anion channel

wt: wild type

wtαΒCry: wild type mice overexpressing αΒ-Crystallin

αMHC: α myosin heavy chain

Δψm: mitochondrial membrane potential

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Introduction

Desmin, the muscle-specific intermediate filament (IF) protein, forms a 3-dimensional scaffold that

interconnects the contractile apparatus to the nucleus, cellular organelles and the sarcolemma

(Capetanaki et al., 2007). The desmin scaffold is a primary target for cardiomyopathy and heart

failure (HF) both in mice and humans (Goldfarb et al., 1998; Capetanaki et al., 2007; Capetanaki et al.,

2015). Mice deficient for desmin develop both skeletal and myocardial defects that are strongly linked

to impaired mitochondrial morphology and function (Milner et al., 1996; Milner et al., 1999; Milner et

al., 2000; Li et al., 1996; Papathanasiou et al., 2015). These mitochondrial abnormalities lead to

cardiomyocyte death and myocardial degeneration, accompanied by inflammation and fibrosis,

resulting in dilated cardiomyopathy (DCM) and HF (Milner et al., 2000; Capetanaki et al., 2007;

Psarras et al., 2012; Mavroidis et al., 2015). In humans, over 70 desmin mutations have been linked to

desmin-related cardiomyopathies, of which DCM is the most frequent (Capetanaki et al., 2015; van

Spaendonck-Zwarts et al., 2011). In addition, desmin aggregate formation due to caspase cleavage is a

major mediator of the TNF-α-induced HF (Panagopoulou et al., 2008). Importantly, regardless of the

specific mutations, human DCM-linked HF displays mitochondrial and other structural abnormalities

similar to both desmin-deficient hearts and TNF-α-induced HF. Furthermore, the R120G mutation of

αB-crystallin, an abundant small heat shock protein, also causes the same desmin-aggregate-related

DCM and HF with similar mitochondrial defects (Wang et al., 2001; Vicart et al., 1998).

Mitochondrial abnormalities seem to be a common feature of diseases in which desmin and/or αB-

crystallin deficiencies are implicated. Previous observations have suggested a physical association of

desmin with mitochondria as well as mitochondrial homeostasis (Stromer et al., 1990; Reipert et al.,

1999; Hnia et al., 2011), but the underlying mechanisms remain elusive. On the other hand, αΒ-

crystallin associates with all three cytoskeletal networks, microfilaments (Bennardini et al., 1992;

Singh et al., 2007), microtubules (Arai et al., 1997; Houck et al., 2010) and IFs (Nicholl et al., 1994;

Iwaki et al., 1989; Wisniewski et al., 1998) [reviewed in (Wettstein et al., 2012)], and specifically

desmin (Bennardini et al., 1992; Nicholl et al., 1994; Perng et al., 1999) and there is, also, evidence

that a fraction of αΒ-crystallin is located in mitochondria (Fountoulakis et al., 2005; Martindale et al.,

2005; Maloyan et al., 2005; Mitra et al., 2013), without its role there being completely understood.

Mitochondria are double membrane-bounded organelles involved in major cellular processes.

Proper assembly, maintenance and function of the various mitochondrial complexes, including

oligomers of OPA1 (optic atrophy 1) (Frezza et al., 2006), F1Fo-ATP synthase (Strauss et al., 2008)

and the recently identified mitochondrial contact site and cristae organizing system (MICOS) (Harner

et al., 2011; Hoppins et al., 2011; von der Malsburg et al., 2011) are vital to inner membrane

organization and the proper and efficient mitochondrial function. Mitofilin (Mic60) (Gieffers et al.,

1997; Odgren et al., 1996), the core component of MICOS, interacts with several inner and outer

mitochondrial membrane (OMM) proteins (von der Malsburg et al., 2011; Darshi et al., 2011; Xie et

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al., 2007; Harner et al., 2011; Hoppins et al., 2011; van der Laan et al., 2012), thus maintaining IMM

morphology and contact sites between IMM and OMM and affecting mitochondrial function, mtDNA

integrity and protein and metabolite trafficking.

To address the role of desmin and αB-crystallin, and their potential interplay in mitochondrial

homeostasis and cardioprotection, we investigated the consequences of αΒ-crystallin overexpression

in the desmin-deficient cardiomyopathy model. We demonstrated that cardiac specific αB-crystallin

overexpression rescues desmin-deficient heart failure and this takes place via maintenance of proper

mitochondrial composition, structure and function, inhibition of abnormal mitochondrial permeability

transition pore (mPTP) activation and cardiomyocyte death prevention. Moreover, we found that

among other sites, both desmin and αΒ-crystallin localize at sarcoplasmic reticulum (SR)-

mitochondria associated membranes (MAMs), where they interact with VDAC. Furthermore, we have

unraveled a potential link between desmin and αB-crystallin with mitochondrial morphology and

bioenergetics through their interaction with Mic60 and ATP synthase.

Results

Generation of des-/- mice overexpressing αΒ-crystallin in the heart

In order to examine the effect of αΒ-crystallin overexpression in the des-/- myocardium, we generated

transgenic mice overexpressing αΒ-crystallin specifically in the heart under the control of the alpha

myosin heavy chain (αMHC) promoter. Among different lines generated, two were chosen for further

investigation, namely αΒCry4 and αΒCry6, with high and low αB-crystallin expression levels

respectively (Fig.1A and S1A). These mice were crossed with des-/- mice to generate αΒ-crystallin

overexpressing des-/- mice (des-/-αΒCry). Quantification of total αB-crystallin protein levels from

hearts of wt, des-/- and transgenic lines revealed a 1.3 fold higher αB-crystallin levels in des-/- mice, a

5.4 fold in line αΒCry4 and a 2.6 fold in line αΒCry6 in comparison to wt.

Overexpression of αΒ-crystallin rescues its lost Z-disc localization due to desmin deficiency and

allows for more efficient mitochondrial localization

It has been shown that, upon stress, αΒ-crystallin translocates from a cytosolic pool to Z-discs

(Golenhofen et al., 1998), where it colocalizes with desmin and α-actinin. Immunofluorescence

microscopy was performed to determine if desmin deficiency would affect the Z-disc localization of

αΒ-crystallin. We found that, in the unstressed wt myocardium, αB-crystallin co-localizes only

partially with desmin and a-actinin at the Z-disc, and is also located at the intercalated discs (IDs)

(Fig.S1B). This localization pattern is enhanced under swimming stress (Fig.1D). In the absence of

desmin, the Z-disc localization of αB-crystallin is mostly lost but its IDs localization is retained

(Fig.1D), possibly due to other potential binding partners at the IDs. Overexpression of αB-crystallin

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in the des-/-αBCry4 myocardium rescues its Z-disc localization (Fig.1D), and more importantly, a

large amount of αB-crystallin is located in mitochondria (Fig.1Bb). Mitochondrial localization of αB-

crystallin also occurs in wt cardiomyocytes but cannot be easily detected. Under swimming stress, the

total protein levels of both desmin and αB-crystallin are increased (Fig.1Ba,Ca,b) and this allows for

easier detection of αB-crystallin in the mitochondrial fraction in both wt and des-/- myocardium

(Fig.1Cc). This is more obvious in the corresponding αB-crystallin overexpressing myocardium

(wtαBCry4 and des-/-αBCry4), however, the ratio of mitochondrial to cytosolic levels remain the

same.

Complete rescue of adverse remodeling in desmin deficient hearts overexpressing αΒ-crystallin

Inflammation, fibrosis and calcification are hallmarks of the des-/- myocardial degeneration and

necrosis (Milner et al., 1996; Mavroidis et al., 2002; Psarras et al., 2012; van Spaendonck-Zwarts et

al., 2011). Dissection of des-/-αΒCry4 mice revealed complete absence of extensive calcification in

the myocardium. Further histological analysis revealed no inflammatory infiltration and minimal

amount of fibrosis in the ventricular walls (Fig.2A-D). Quantification of histological section images

confirmed the significant reduction of fibrosis in the des-/-αΒCry myocardium (Fig.2C). Comparison

of the two transgenic lines, des-/-αΒCry4 and des-/-αΒCry6, revealed that 5.4 fold overexpression of

αB-crystallin is more beneficial when compared to 2.6 fold overexpression. Therefore, the subsequent

studies were done using line des-/-αΒCry4, designated from now on as des-/-αΒCry.

Evans blue dye (EBD) analysis was performed to detect cellular membrane permeability. Wild type

cardiomyocytes were impermeable to EBD since their membranes are intact (Fig.2E,F). In contrast,

des-/- hearts appear to have considerably large areas of EBD positive cells, especially in regions with

inflammatory infiltration. In des-/-αBCry myocardium there were no EBD positive cardiomyocytes;

leading to the conclusion that αB-crystallin overexpression protects des-/- myocardium from necrotic

cell death.

αΒ-crystallin overexpression considerably improves cardiac function and results in 100%

survival during obligatory swimming exercise

Heart failure development with aging is characteristic of des-/- hearts. To explore the effect of αΒ-

crystallin overexpression on cardiac function, we performed standard morphometric analysis and 2D-

directed M-mode echocardiography (Fig.3 and Table S1). Des-/-αBCry mice show a significant

improvement in all parameters tested in comparison to des-/- mice. The improvement in left

ventricular (LV) function of des-/-αBCry mice is very extensive, reaching almost wt levels (96% of

wt fractional shortening) (Fig.3Bc), consistent with the improvement in LV-end systolic diameter

which was also comparable to wt levels (Fig.3Bb). Posterior wall thickness was also increased by

35% in the des-/-αBCry compared to the des-/- group, consistent with the absence of degeneration in

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these hearts (Fig.3Bd). Finally, the ratio of LV radius to PWT, an indicator of LV wall stress, was

significantly lower in des-/-αBCry mice compared to des-/- (Fig.3Be).

In order to investigate whether this cardioprotection can also take place under stress conditions, wt,

des-/- and des-/-αΒCry mice participated in an obligatory swimming exercise protocol for 24 days.

The des-/- mice have considerably reduced ability to handle stress as 50% of them die during

swimming (in comparison to 0% of wt) (Fig.3C). The survival rate of des-/-αΒCry mice was 100%,

strongly demonstrating the powerful cardioprotective action of αΒ-crystallin overexpression against

stress.

Rescue of des-/- cardiomyocyte ultrastructural defects by αΒ-crystallin overexpression

In an effort to understand the mechanism by which the overexpression of αΒ-crystallin protects des-/-

myocardium against degeneration and improves cardiac function, we initially examined its effects on

cardiomyocyte ultrastructure. Des-/- cardiomyocytes are characterized by various early appearing

mitochondrial defects (Milner et al., 2000), followed by other structural abnormalities and loss of

muscle integrity (Fig.3D). Mitochondrial abnormalities include disturbed shape and positioning,

fragmentation, aggregation, swelling of the mitochondrial matrix, disruption and fragmentation of the

cristae and degeneration (Fig.3E). In contrast, none of these ultrastructural defects are present in des-/-

αΒCry mice. Myofibrils are more properly aligned and mitochondria, located parallel to myofibrils,

appear normal, without signs of fragmentation, cristae abnormalities or destruction. These data

suggest that mitochondria are a major target of αΒ-crystallin cytoprotection.

αΒ-Crystallin overexpression provides anti-oxidant protection to des-/- cardiomyocytes

To further investigate the mechanism by which αΒ-crystallin protects against mitochondrial defects

we analyzed its effects on adult cardiomyocyte reactive oxygen species (ROS) levels using the

molecular probe CM-H2DCFDA (Fig.4A,B). Des-/- cardiomyocytes demonstrate a significant higher

degree of ROS over the wt levels, while des-/-αΒCry cardiomyocytes are characterized by a lower

degree of ROS. Under oxidative stress conditions, simulated by incubation with H2O2, there is

significant decrease of ROS levels in both des-/-αΒCry and wtαBCry compared to des-/- and wt cells

respectively, indicative of the protection provided by αΒ-crystallin overexpression. To further

investigate the mechanism of anti-oxidant protection by αΒ-crystallin we measured the levels of

glutathione. In des-/- hearts glutathione levels are decreased by 33% and GSH/GSSG ratio by almost

50% in comparison to wt (Fig.4C). Overexpression of αΒ-crystallin increased glutathione and

GSH/GSSG levels to 87% and 95% respectively of wt levels suggesting that, at least partially, the

mechanism of anti-oxidant protection by αΒ-crystallin takes place via an increase of reduced

glutathione levels.

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αΒ-crystallin overexpression inhibits abnormal activation of the mitochondrial permeability

transition pore (mPTP) and the dissipation of mitochondrial membrane potential (Δψ)

Mitochondrial swelling and increased ROS levels have been linked to abnormal activation of the

mPTP. Therefore, we investigated whether the above demonstrated mitoprotection and

cardioprotection by αΒ-crystallin overexpression takes place through inhibition of mPTP activation.

First, we determined the sensitivity of isolated mitochondria to increased Ca2+ levels in the presence

or absence of the mPTP inhibitor Cyclosporin A (CsA). As expected, des-/- mitochondria swell more

than wt mitochondria at the same Ca2+ concentration, as measured by the decrease in absorbance at

540 nm (Table S2). On the other hand, des-/-αBCry mitochondria showed a significant decrease in

swelling, indicating that overexpression of αB-crystallin improves the diminished capacity of des-/-

mitochondria to resist exposure to Ca2+. Addition of CsA before the Ca2+challenging significantly

abrogated mitochondrial swelling. This finding demonstrates that first, the reduced ability of des-/-

mitochondria to resist increased Ca2+ levels (Weisleder et al., 2004) is mPTP dependent and second,

the mechanism of mitoprotection and inhibition of necrotic cell death of des-/- myocardium by αB-

crystallin overexpression involves suppression of mPTP activation.

In the des-/- heart, mitochondrial abnormalities, including swelling, can lead to dissipation of Δψm, a

critical event in the process of cell death. To address this possibility, we incubated adult

cardiomyocytes with tetramethylrhodamine methyl ester (TMRM), a potentiometric indicator that is

targeted to mitochondria. TMRM intensity was 21% lower in des-/- cardiomyocytes compared to wt,

indicating reduced Δψm (Fig.4D,E). Time-lapse confocal scanning showed a more rapid loss of

TMRM in des-/- relative to wt mitochondria, consistent with mitochondrial membrane depolarization

(Fig.4F and S2). αB-crystallin overexpression in des-/- myocardium significantly increased both the

intensity and retention time of TMRM. To examine whether the accelerated TMRM loss in des-/-

cardiomyocytes was due to mPTP activation, we treated cardiomyocytes with CsA, significantly

attenuating TMRM loss.

αΒ-crystallin overexpression restores changes in the levels of important mitochondrial proteins

caused by desmin deficiency

In an effort to understand the underlying mechanisms responsible for both the observed mitochondrial

defects in the absence of desmin and most importantly the unprecedented extensive rescue of these

defects by αΒ-crystallin overexpression, we conducted proteomic analysis to identify any critical

alterations in the mitochondrial proteome of the different genotypes. The results revealed diminished

levels of enzymes involved in the citric acid cycle, lipid metabolism and fatty acid beta-oxidation in

the des-/- compared to wt mitochondria (Table 1 and S3, Fig.S3). Moreover, in des-/- mitochondria

the levels of many subunits and components of the electron transport chain were decreased, along

with components of complexes linking different metabolic pathways. The most extensive change was

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found with the cristae junction protein Mic60 (IMMT), which was decreased approximately four fold

(Table 1, Fig.5A,B). On the contrary, in the mitochondria of des-/-αΒCry myocardium, most of the

diminished protein levels, including that of Mic60 protein, were apparently restored (Table 1 and S4,

Fig.5). In agreement with the WB data (Fig.1B), proteomic analysis revealed that much higher levels

of αΒ-crystallin localizes to the mitochondria of the des-/-αΒCry hearts in comparison to des-/- hearts.

The levels of some of the differentially expressed proteins were also evaluated by WB analysis

(Fig.5A,B). In addition, we investigated the mitochondrial levels of ANT (adenine nucleotide

translocator), an IMM protein with known mitochondrial and cardiomyocyte function (Narula et al.,

2011; Vogelpohl et al., 2011) that cannot be easily detected by proteomic analysis. This analysis

revealed that, in contrast to total ANT levels, mitochondrial ANT levels are reduced in des-/-

myocardium, a defect ameliorated by αB-crystallin overexpression (Fig.5A, B).

Desmin and αΒ-crystallin interact with proteins important for cristae morphology

As demonstrated above, one of the most interesting proteins downregulated in des-/- mitochondria is

Mic60 (Table 1, Fig.5A), consistent with the shown cristae morphology defects (Fig.3D,E). Given

that both these defects are rescued by αΒ-crystallin overexpression, we aimed to further unravel the

underlying mechanism. We used GST-tagged desmin and co-immunoprecipitation to examine

whether desmin interacts with Mic60, and therefore, that loss of this interaction in des-/-

cardiomyocytes affects its proper mitochondrial targeting and/or stabilization. Indeed, we found that

desmin interacts with Mic60, and as expected, with αΒ-crystallin (Fig.5C,D). More importantly, we

demonstrated that αΒ-crystallin also associates with Mic60 (Fig.5D), both in wt and des-/- myocardial

extracts, strongly suggesting that Mic60 is one of αΒ-crystallin targets in the des-/- mitochondria.

Using similar immunoprecipitation studies, (and immunoprecipitation coupled to proteomic analysis

for the case of desmin), we found that the beta subunit of the F1 catalytic domain of ATP synthase,

another protein important for determining cristae structure (Paumard et al., 2002) and mitochondrial

function (Zick et al., 2009), interacts with desmin as well as with αΒ-crystallin (Fig.5C,D). Loss of

this interaction in the absence of desmin, combined with the disrupted cristae and depolarized Δψm, is

expected to impact the ATP content, which we indeed confirmed to be 40% lower in the des-/-

cardiomyocytes compared to wt (Fig.5E). αΒ-crystallin overexpression increased this ATP content to

90% of the wt levels.

These findings support the view that the loss of desmin association with these proteins may be a key

mediator of the des-/- pathology. Given that αΒ-crystallin also associates with these proteins, its

higher levels in the des-/-αΒCry cardiomyocytes, in comparison to its basal levels in des-/-, may be

able to compensate for the impact of desmin loss to Mic60 and ATP synthase function.

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Desmin and αB-crystallin are located at the SR and MAMs where they associate to VDAC

The question raised by the amelioration of des-/- mitochondrial pathology by αB-crystallin

overexpression is how desmin and αB-crystallin, two mainly cytoplasmic proteins, can interact with

IMM proteins and impact mitochondrial homeostasis. To address this question we first performed

protease accessibility assays to examine whether a fraction of desmin and αB-crystallin is located

inside the mitochondria. Crude mitochondria from wt hearts were isolated and digested with

proteinase K. As shown in figure 6A, both desmin and αB-crystallin are degraded by proteinase K,

while the IMM protein ANT and the OMM protein VDAC remain intact. The digestion pattern of

desmin and αB-crystallin resembles that of mitofusin 2, an OMM protein, as detected by an antibody

that recognizes a part of mitofusin 2 exposed to the cytosol. In contrast, overexpressed αB-crystallin

appears to localize inside mitochondria (Fig.6Ab).

Next, we performed subcellular fractionation of rat and mouse hearts to further determine the

localization of desmin. Interestingly, while we found desmin in the SR fraction, it was mainly

enriched in the SR-mitochondria associated membranes (Fig.6B). In contrast, αB-crystallin was found

enriched more in the SR fraction and less in the MAMs and pure mitochondrial fraction. On the other

hand, overexpressed αB-crystallin was also enriched in the SR fraction but even more so in the

MAMs and in pure mitochondria (Fig.6C). The localization of desmin was confirmed by immunogold

labeling studies (Fig.6D and S4). In addition to the expected places such as IDs (Fig.S4) and Z-lines,

desmin is found to associate with mitochondria and membranous structures, most likely SR, in close

proximity to mitochondria.

A previous yeast two-hybrid screening of a heart cDNA library using the entire desmin molecule as

bait, conducted in our lab, had shown binding of desmin to VDAC (unpublished results). In addition

to its mitochondrial localization, VDAC is also enriched in MAMs, and furthermore it was found to

interact with Mic60 (Hoppins et al., 2011). Therefore, we reconfirmed the binding of VDAC to

desmin using both co-immunoprecipitation studies as well as GST pull-down assays (Fig.7A,B). To

determine the corresponding binding regions of VDAC and desmin, we generated several deletion

constructs and used them in reciprocal GST pull-down experiments. As demonstrated in fig. 7D, the

N-terminal domain of VDAC is necessary for its binding to desmin, while the C-terminal tail domain

of desmin and to a lesser extent the rod domain, are sufficient for association of desmin with VDAC.

The finding of desmin-VDAC binding was further strengthened by double immunogold labeling

studies, which confirmed the close proximity of these two proteins in vivo (Fig.6D and S4). αB-

crystallin was also co-immunoprecipitated with VDAC, consistent with earlier reports (Mitra et al.,

2013). The demonstrated associations of desmin and αB-crystallin with VDAC, Mic60 and ATP

synthase could somehow facilitate the formation and/or stabilization of a scaffold-like supper-

complex extending from ER-mitochondria contact sites to MICOS and ATP complexes.

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αB-crystallin restores macromolecular complexes of Mic60 affected by desmin deficiency

We speculated that desmin and αB-crystallin association with Mic60 could affect the formation or

stabilization of Mic60 containing complexes. We analyzed crude mitochondria from mouse heart

solubilized with digitonin by blue native–PAGE in the first dimension and SDS-PAGE in the second

dimension, followed by detection of Mic60 by WB analysis (Fig.7E,F). In the absence of desmin, the

number of the protein complexes containing Mic60 is much lower compared to wt, consistent with the

diminished Mic60 protein levels found in des-/- mitochondria (Table 1, Fig.5A). Importantly, as

determined by using much lower protein levels, the higher molecular mass complexes of Mic60 are

proportionally less than the corresponding lower molecular mass complexes, indicating a possible

facilitating role of desmin in the formation or stabilization of these complexes (Fig.7F and S4). All

these data are consistent with the cristae defects of des-/- mitochondria (Fig.3). Overexpression of αB-

crystallin in the absence of desmin restores the abundance of all the different size Mic60 complexes to

their normal levels. Importantly, desmin and αB-crystallin appear to be in the same complexes with

Mic60 and VDAC, supporting the idea of a functional role in the interface of SR-mitochondria outer

and inner membranes. Under swimming stress conditions, we obtained similar results with only a

slight shift of VDAC complexes, as observed in the des-/- mitochondria (Fig.7F). This potentially

reflects a partial dissociation of Mic60 and VDAC complexes.

Discussion

Mitoprotection as a major mechanism of αΒ-crystallin-mediated cardioprotection in desmin deficient

heart failure

We have demonstrated that αB-crystallin overexpression in the des-/- myocardium completely

halts the development of heart failure. The present results show that, while its mechanism of

cardioprotection may be occurring at various levels, αB-crystallin overexpression definitively

provides extensive mitoprotection to des-/- cardiomyocytes. The earliest features of the des-/-

pathology are mitochondrial structural perturbations, evident as early as the second week after birth,

before any other cardiac dysfunction arises (Milner et al., 2000). These findings are further supported

by additional studies showing that overexpression of Bcl-2, which has a known protective action on

mitochondria, provides significant improvement of des-/- cardiomyopathy (Weisleder et al., 2004).

Our studies demonstrated an increase in oxidative stress, an increased activation of mPTP, and

decreased Δψm in the absence of desmin. All these mitochondrial defects are corrected in the des-/-

αBCry myocardium, in which mitochondria have an apparently wt phenotype. It has been shown that

the αB-crystallin mutation R120G, known to cause desminopathy characterized by desmin and αB-

crystallin aggregation, also leads to redox imbalance (Rajasekaran et al., 2007). Defining the extend

by which these aggregates contribute to the development and progression of the disease is of great

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importance. Clearing away the aggregates improves this model of proteotoxicity (Li et al., 2011; Su et

al., 2015; Cabet et al., 2015; Pattison et al., 2011; Gupta et al., 2014), but it does not compensate for

the loss of function of the aggregated proteins. Previous and present data with the des-/- model, which

has no desmin and αB-Crystallin aggregates, but displays similar cellular and functional defects with

those of desmin/αB-Crystallin related cardiomyopathy (Milner et al., 1996; Milner et al., 1999; Milner

et al., 2000; Li et al., 1996; Pattison et al., 2011; Wang et al., 2003; Wang et al., 2006; McLendon et

al., 2011), suggest alternate causes for this pathology, in addition to the gain of toxic function

(Bhuiyan et al., 2013).

αB-crystallin and Desmin: old partners in a new game

The association of desmin with αΒ-crystallin, and the fact that mutations in either one of them

lead to heart failure with common characteristics, suggest a potential compensatory interplay between

these two proteins in mitoprotection and consequently cardioprotection. Indeed, we found that both

proteins are protecting mitochondria by, among other possible mechanisms, maintaining proper

composition of mitochondrial proteins. This could take place either by facilitating efficient protein

targeting and transport of the nuclear encoded mitochondrial proteins into the mitochondria, or by

helping in the formation and/or stabilization of mitochondrial protein complexes. Up to now, the

former possibility was considered more probable for a desmin-based scaffold, which, although it does

associate to mitochondria via plectin (Winter et al., 2008), is mainly cytosolic. The present study

demonstrates a novel way by which desmin can regulate mitochondrial homeostasis. The contact sites

where SR is linked to the mitochondrial outer and inner membranes, are established sites for multiple

cellular processes, including Ca2+ and metabolite transfer, lipid metabolism, mitochondrial shape

regulation, and autophagosome and inflammasome formation (Naon et al., 2014). The desmin

scaffold, localized at these contact sites, could contribute to all of the above processes, and

particularly to the maintenance of SR-mitochondria proximity. This proximity allows the desmin

scaffold, together with αΒ-crystallin, to facilitate directed protein and metabolite targeting to

mitochondria. As shown here, and was previously suggested by others (Fountoulakis et al., 2005;

Martindale et al., 2005; Maloyan et al., 2005), αB-crystallin does localize in and outside mitochondria,

and thus, could support multiple processes. The potential compensatory interplay between desmin and

αΒ-crystallin is further supported by the fact that both of them associate with important mitochondrial

proteins. Our data indicate that desmin plays a possible role in the formation/stabilization of Mic60

macromolecular complexes. In the absence of desmin this role is restored by overexpressed αB-

crystallin. As discussed above, Mic60 is crucial for the formation and function of the extended cristae

membrane structures, the main sites of ATP synthesis by oxidative phosphorylation (Gilkerson et al.,

2003; Vogel et al., 2006; Wurm et al., 2006). In addition, it is located at the contact sites (Harner et al.,

2011) and has recently been implicated in mitochondrial protein import (van der Laan et al., 2012). It

is thus conceivable that Mic60 decrease in the absence of desmin could compromise protein import

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into mitochondria. Our proteomic analysis revealed that the levels of many enzymes involved in

aerobic respiration are diminished in the des-/- cardiomyocytes (Table 1,S3,S4), while αΒ-crystallin

overexpression restores them, thus conferring to the cell high-energy electrons in the form of NADH

and FADH2, which can be used for ATP generation by oxidative phosphorylation.

In addition to Mic60, F1F0-ATP synthase affects cristae membrane structure by imposing

curvature through dimerization and oligomerization (Strauss et al., 2008; Dudkina et al., 2006;

Minauro-Sanmiguel et al., 2005), thus providing the required conditions to enhance the catalytic

capacity of the OXPHOS system (Strauss et al., 2008). Recently, mitochondrial cristae shape was

found to determine respiratory chain supercomplex assembly and respiratory efficiency (Cogliati et al.,

2013). Such efficiency minimizes the downstream ROS production discussed above, consistent with

the proposed upstream action of αΒ-crystallin. All these data are also consistent with the significant

decrease of ATP levels in des-/- hearts and their restoration by αΒ-crystallin overexpression.

Moreover, recent work indicated that mPTP can be formed from ATP synthase dimers (Giorgio et al.,

2013). The interaction of desmin and αΒ-crystallin with both ATP synthase and VDAC suggests a

possible role of these proteins in the regulation of mitochondrial bioenergetics and mPTP opening.

The present data suggest that the desmin cytoskeletal network could facilitate the

mitoprotective/cardioprotective role of αB-crystallin in its baseline expression levels. In the absence

of desmin, αB-crystallin loses its Z-disc localization and thus the ability to reach the required high

concentrations at a Z-disc proximal SR/ER-mitochondrial site for its proper function. Indeed, much

higher levels of αB-crystallin are required to facilitate the same level of protection in the absence of

desmin. This is consistent with the presently proposed involvement of both proteins in the ER-

mitochondria organizing network, which might be only part of the action of these partners on

mitochondrial behavior.

Materials and methods

Transgenic animal generation

Des-/- mice were previously generated in the 129SV inbred background (Milner et al., 1996).

Transgenic mice overexpressing αΒ-crystallin were generated in C57BL/6J background, using the

aMHC promoter (Genbank U71441) (Subramaniam et al., 1991), driving expression of a full length

murine αΒ-Crystallin cDNA, followed by the bovine growth hormone poly-A sequence and

backcrossed onto pure 129SV mouse strain for several generations. The animals were used

independently of sex. The approved procedures for animal care and treatment were according to

institutional guidelines following those of the Association for the Assessment and Accreditation of

Laboratory Animal Care (AAALAC) and the recommendations of Federation of European Laboratory

Animal Science Association (FELASA).

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Southern and Western blot

For the identification of transgenes by southern blotting, a fragment of the αMHC was isolated and

used to generate 32P-labeled probes by random priming.

For WB analysis, proteins were transferred to polyvinylidene fluoride membrane (Whatman) and

probed with the following antibodies: anti-αB-Crystallin (Stressgen: SPA223- 1:2000; Santa Cruz: sc-

22744-1:1000), anti-desmin (abcam: ab8592-1:1000), anti-GAPDH (Ambion: AM4300-1:4000), anti-

ANT (Santa Cruz: sc-9299-1:200), anti-ATP5B (Mitosciences: ab5432-1:500), anti-mitofilin (Novus

Biologicals: NB100-1919-1:1000; abcam: ab110329- 1:1000), anti-NDUFS2 (Thermo Scientific:

PA5-19342-1:500), anti-SDHA (ab14715-1:1000), anti-COXIV (ab33985-1:1000), anti-VDAC1

(ab14734-1:1000; ab15895-1:1000) (all abcam), anti-mitofusin 2 (Sigma: M6444-1:1000), anti-

MnSOD (BD Biosciences:M9920-1:500). Secondary HRP-conjugated antibodies were purchased

from Sigma. Proteins were visualized using chemiluminescence (ECL kit, Amersham). Films were

scanned using a GS800 densitometer (Bio-Rad) and quantified using Quantity one software (Bio-Rad).

Histology

For the evaluation of fibrosis, Masson’s trichrome stained paraffin sections were photographed and

subjected to Image J software analysis. The results were expressed as percentage of fibrosis in a given

tissue section. For the evaluation of inflammation, hearts were observed under a Stemi 2000-C

stereomicroscope (Zeiss) and were graded from 0-4, according to the presence of inflammatory

infiltrate, as previously described (Psarras et al., 2012).

Evans Blue Staining

The mice were injected intraperitoneally with Evans Blue Dye (250μg/g of body weight in 1xPBS).

After 24h, the heart was embedded in O.C.T compound (VWR) and snap frozen in liquid nitrogen.

EBD staining was evaluated by the Image J software and the results were expressed as percentage of

EBD in the area imaged.

Electron Microscopy

Mice were treated with heparin for 30min, sacrificed and the heart was perfused with 2,5%

Glutaraldehyde in 0.1 M Phosphate Buffer (PB), pH 7.4, fixed overnight and post-fixed with 1%

osmium tetroxide for 1h at 4°C, dehydrated, embedded in Epon/Araldite resin mixture and allowed to

polymerize at 60°C for 24h. Ultrathin sections (65-70 nm) (Leica EM-UC7 ultramicrotome, Leica)

were examined with a Philips 201C transmission electron microscope and photographed with Agfa

Copex HDP13 microfilm.

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For immunogold labeling, mice were perfused with cold 4% paraformaldehyde and 1%

glutaraldehyde in 0.1 M PB, pH 7.4. Hearts were post-fixed in the same fixative for 2h and rinsed in

0.1 M PB. The dehydration and embedding of tissue samples was carried out with the PLT method

using a Leica EM AFS (Leica) according to (Robertson et al., 1992).

Double immunogold labeling

Mounted ultrathin sections were submitted to the postembedding immunogold procedure according to

(Havaki et al., 2003; Diokmetzidou et al., 2016). All incubations were carried out using the automated

immunogold labeling system Leica EM IGL. Primary antibodies (VDAC: abcam-ab147234-1:10,

desmin: abcam-ab8592-1:20) were incubated overnight at 4°C and the secondary anti-polyclonal IgG-

10 nm gold particles [1:40] and anti-monoclonal IgG-25 nm gold particles [1:40] for 1h at RT.

Sections were examined with a Philips 420 transmission electron microscope at 60 kV and

photographed with a Megaview G2 CCD camera (Olympus SIS).

Immunofluorescence

Frozen mouse cardiac sections were used for immunolabeling as previously described (Diokmetzidou

et al., 2016). Primary antibodies against αB-crystallin (Stressgen: 1:100, Santa Cruz: 1:50) and anti-α-

actinin (Sigma: Α7811-1:1000) were incubated overnight at 4°C and secondary (Alexaflour-594 and

Alexaflour-488, Invitrogen) for 1h at RT. The sections were examined using a Leica, TCS SP5

confocal microscope equipped with a HC-PL-APO-CS-63x OIL objective and LAS-AF acquisition

software (Leica).

Swimming trial

Mice were exercised twice a day using a swimming protocol as previously described (Milner et al.,

1999). The swimming program began at 10 min/day and was increased by 10 min daily until a

maximum of 1h. The double 1h exercise was continued for nine days and subsequently was increased

to 90 min. The double 90 min exercise was continued for 9 days. In case of αB-Crystallin localization

studies, a three day protocol was followed.

Echocardiography

Mice were anesthetized with intraperitoneal injection of ketamine (100 mg/kg). Echocardiographic

studies were performed using a Vivid 7, GE ultrasound system with a 13 MHz linear transducer. Two-

dimensional targeted M-mode imaging was obtained from the short axis view at the level of greatest

LV dimension. Images were analyzed using the Echopac PC SW 3.1.3/ software (GE). Data were

expressed as mean±s.d. for continuous variables and analysed by using Statview 5.0 (Abacus

Concepts). Statistical comparisons were performed using ANOVA with Bonferroni/Dunn post-hoc

test or the unpaired Student’s t-test where appropriate.

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Adult cardiomyocyte isolation

Isolation was performed using a modified protocol (Diokmetzidou et al., 2016) based on standard

procedures (O'Connell et al., 2007).

Measurement of mitochondrial membrane potential (Δψm), mPTP opening and ROS

Adult cardiomyocytes were incubated with 20 nM TMRM (Invitrogen) for 15 min at 37oC, washed

and examined by time lapse confocal scanning for 20 min with 1 min interval (TCS SP5, Leica) by

excitation at 568 nm and emission at 580-640 nm. 5 μM cyclosporine A (Invitrogen) was added with

TMRM to confirm mPTP opening. For the ROS measurement, the cardiomyocytes were incubated

with 20μΜ CM-H2DCFDA (Invitrogen) for 30 min at 37oC, washed and examined using a 488 nm

argon-ion laser excitation and emission at 503-555 nm. A HCX-PL-APO-CS-20x0,70 DRY UV

objective and LAS-AF acquisition software (Leica) were used in both cases. The acquired images

were analyzed using the Volocity 5 software (Perkin Elmer).

Ca2+-induced swelling of mitochondria was performed as previously described (Weisleder et al.,

2004). 5 μΜ CsA were added to mitochondria 5 min before CaCl2.

Tissue fractionation, mitochondrial purification and mitochondria-associated membranes

isolation

Fractionation of mouse or rat hearts and the isolation of mitochondria-associated membranes and pure

mitochondria were performed by differential centrifugation at 4oC as previously described in

(Wieckowski et al., 2009; Diokmetzidou et al., 2016; Frezza et al., 2007).

Glutathione measurements

Glutathione concentration was measured in hearts of 6-month-old mice as previously described

(Rahman et al., 2006). The hearts were weighted, homogenized in 5% sulfosalicilic acid and

centrifuged at 10000 xg for 10 min at 4°C. The supernatant was removed and used for the GSH assay

based on (Griffith, 1980).

Determination of ATP levels

ATP content was measured using the Aposensor ATP assay kit (Biovision) in a luminometer

(Berthold) according to the manufacturer’s instructions.

Two-dimensional blue native/SDS PAGE

Mouse cardiac mitochondria were solubilized in native solubilization buffer pH 7 (20 mM Bis-Tris,

500 mM aminocaproic acid, 20 mM NaCl, 2 mM EDTA pH8, 10% glycerol, 2 mM

phenylmethylsulfonyl fluoride, protease inhibitor cocktail) with 4 g digitonin per g of protein for 30

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min on ice and analyzed in a 3-12% native gel (Serva). Individual lanes were excised, incubated in

equilibration buffer (125 mM Tris-HCl, pH 6,8 and 1% SDS) for 20 min and analyzed at the second

dimension by 10% gel SDS-PAGE. Proteins were transferred to PVDF membrane (Whatman) and

probed with the desired antibodies.

Two-dimensional gel electrophoresis (2DE)

2DE was performed as previously described (Makridakis et al., 2010) with the following

specifications: mitochondrial fractions were solubilized in the IEF sample buffer (7 M Urea, 2 M

Thiourea, 4% CHAPS, 1% DTE, 2% IPG) and resolved (100 μg per sample) on 7 cm strips pH range

3-10 Non Linear (Bio-Rad) using the in gel rehydration method. IEF was conducted for about

12500Vhrs. Strips were then incubated in equilibration buffer (6 M Urea, 50 mM Tris-HCl pH 8.8,

30% Glycerol, 2% SDS) containing 0.5% DTE for 45 min with light agitation followed by a second

incubation with equilibration buffer containing 4.32% IAM. Second dimensional analysis was

performed on 12% SDS-PAGE and the gels were stained with Coomassie Colloidal Blue stain

(Candiano et al., 2004).

Spot quantification

Gels were scanned at a GS-800 imaging densitometer (Bio-Rad) in transmission mode and image

analysis was performed with the PD Quest 8 software package (Bio-Rad). Individual protein spot

quantity was normalized based on the total quantity of the spots in the gel and expressed in ppm.

Statistical analysis was conducted by the use of Mann-Whitney and Student’s t-test.

Protein Identification via MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization-Time

of Flight Mass Spectrometry)

Protein spots were excised manually or automatically by the use of the ProteineerSp Protein picker

(BrukerDaltonics), destained (30% Acetonitrile, 50 mM NH4HCO3), washed, dried and trypsinized

o/n (trypsin proteomics grade Roche). Peptides were extracted (50% Acetonitrile, 0.1% TFA) and

applied on MALDI target with the dry droplet method. Peptide masses were determined by MALDI-

TOF/TOF MS (Ultraflex TOF/TOF, BrukerDaltonics), peak list was created with Flexanalysis v3.3

software (Bruker), smoothing was applied with Savitzky-Golay algorithm (width 0.2 m/z, cycle

number 1), and a signal/noise threshold ratio of 2.5 was allowed. For peptide matching (Mascot

Server 2; Matrix Science), the following settings were used: monoisotopic mass, one miscleavage site,

carbamidomethylation of cysteine as fixed and oxidation of methionine as variable modifications.

Stringent criteria were used for protein identification with a maximum allowed mass error of 25 ppm

and a minimum of 4 matching peptides. False identity probability was usually lower than 10-5. Data

analyzed with the same settings as described above, using a sequence scrambled version of Swiss-Prot

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data base generated by the decoy generating-script available at Matrix Science, provided no

identifications.

Expression and purification of GST tagged proteins

Desmin mutants: Head: 1-108aa (1-324 bp), Head-Rod: 1-412aa (1-1236 bp), Rod: 109-412aa (324-

1236bp), Rod-Tail: 109-470aa (324-1410 bp), Tai: 413-470aa (1237-1410bp) and VDAC ORF and

mutants: ΔNterm: 33-296aa (99-888 bp) lacking the N-terminus α-helix and Δβ1-β4 which lacks 34-

81aa (102-243 bp) thus missing barrels 1-4, were PCR isolated using wt mouse heart cDNA as

template and DNA Q5 High Fidelity polymerase and sub-cloned into pGEX-4T-3. The recombinant

polypeptides were expressed into BL21 bacteria and the proteins isolated as previously described

(Kouloumenta et al., 2007; Diokmetzidou et al., 2016)

GST pull-down and co-immunoprecipitation

Five mg mouse cardiac mitochondria were solubilized in native buffer pH 7 (20 mM Bis-Tris, 500

mM aminocaproic acid, 20 mM NaCl, 2 mM EDTA pH 8, 10% glycerol, 2 mM PMSF, protease

inhibitor cocktail), containing 4 g digitonin/g of protein, for 30 min on ice, and precleared for 3h at

4oC and used according to (Diokmetzidou et al., 2016). The bound proteins were eluted by heating at

97oC for 10 min in 2x electrophoresis loading buffer or in the case of GST pull-down with 20 mM

reduced glutathione in 50 mM Tris-HCl, pH 8, 30 min at RT. The eluates were analyzed by WB.

Protease accessibility assay

One hundred μg (1 mg/ml) of crude mouse cardiac mitochondria were suspended in MSE (225 mM

mannitol, 75 mM sucrose, 1 mm EGTA, 1 mM Tris-HCl pH7,4) or osmotic buffer (20 mM KCl) with

or without 0,5% Triton X-100 and incubated with 2 μg/ml proteinase K for 1h on ice. The reaction

was stopped with 4 mM (final) PMSF. Samples were loaded with 2x SDS-PAGE loading buffer and

analyzed by WB.

Statistical analysis

All data are expressed as mean±s.e.m. unless otherwise stated. Statistical comparisons were

performed using ANOVA with Bonferroni/Dunn post-hoc test or unpaired Student’s t-test where

appropriate. A p value <0.05 was considered significant. Graphics and statistical analysis were

performed using Sigma Plot 10.0 (Systat Software).

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Acknowledgements

We thank very much S. Pagkakis and E. Rigana for their help with imaging. We are grateful to E.

Mavroidis and I. Kostavasili for constant assistance throughout this work. We also thank prof.

William James Craigen and N. Flytzanis for valuable comments on the manuscript.

Competing interest

No competing interests declared.

Author Contribution

AD designed and performed experiments, analyzed data and wrote the manuscript; ES, MT,

performed experiments; IK performed the electron microscopy experiments; MM and AVl performed

and analyzed the proteomic experiments SG helped with transgenic mice generation, AV and CHD

performed and analyzed the echocardiography; YC directed the research project, analyzed the data

and wrote the manuscript.

Funding

This work was supported by PENED 01ED371/Onassio Cardiac Surgery Center, EPAN YB-22 and

PEP ATT-39 and ESPA 09SYN-21- 965 and ‘’Excellence II’’/ARISTEIA II 5342 grants from the

Greek Secretariat for R&D to YC and in part by a FP7/REGPOT-2008-1 (Transmed) to AV.

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Figures

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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 wt, des-/- and αΒ-

crystallin overexpressing (αBCry4 and αBCry6) transgenic mice. GAPDH (glyceraldehyde 3-

phosphate dehydrogenase): loading control. (B) WB analysis of total heart lysates (a) and subcellular

fractions (b) from mice in control conditions and after a swimming exercise reveals extended

localization of αΒ-crystallin in the mitochondria of αBCry4 hearts; GAPDH: cytosolic marker,

COXIV (cytochrome C oxidase IV): mitochondrial marker. The arrow indicates nonspecific bands. (C)

Cardiac protein levels of (a) desmin, (b) αB-crystallin in normal and stressed conditions and (c) the

protein levels of αB-crystallin located in the cytosol and mitochondria. Error bars show mean±s.e.m.

(a) ***p<0.001 vs wt control, *p≤0.05 vs wtαBCry control; (b) **p<0.001 vs wt control,

***p<0.0001 vs des-/- control and swimming, *p<0.05 vs des-/-αBCry control. (D) Double

immunofluorescence staining with αΒ-crystallin and α-actinin antibodies of frozen myocardial tissue

sections from 6-month-old mice (αBCry4), fixed right after swimming exercise (blue staining of

nuclei: DAPI, arrows: IDs, scale bar: 10 μm).

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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 wt

(n=7), des-/- (n=7), des-/-αBCry4 (n=10) and des-/-αBCry6 (n=7) mice. Fibrosis is detected by blue

staining of collagen deposition. (B) Hematoxylin and eosin staining of 23-day-old wt (n=6), des-/-

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(n=12), des-/-αΒCry (n=15) and wtαΒCry (n=6) mice, showing the inflammatory infiltration in the

des-/- myocardium compared to the others genotypes; scale bar: 20 µM. (C) Semi-quantitative

determination of myocardial fibrosis by Image J software analysis **p<0.01 vs wt and des-/-αBCry6,

***p<0.0001 vs des-/-, #p<0.001 vs wt and des-/-αBCry4. (D) Assessment of cardiac inflammation,

using 23-days-old hearts, by a conventional grade ***p<0.0001 vs wt, des-/-αΒCry and wtαΒCry. (Ε)

Evaluation of cardiomyocyte membrane permeability by Evan’s blue (red) and a-actinin (green)

staining of frozen heart sections from 23-day-old mice; blue staining of nuclei: DAPI, scale bar: 100

µm. (F) Quantification of EBD positive area by Image J software analysis, p<0.0001. All error bars

show mean±s.e.m.

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Fig. 3. αΒ-crystallin overexpression improves considerably cardiac function, survival rate

during obligatory exercise and des-/- cardiomyocyte ultrastructural defects. 7-month-old mice

were subjected to echocardiography. Des-/-αBCry mice show a significant improvement in left

ventricular (LV) function reaching FS to 96% of wt levels. (A) Representative M-mode

echocardiogram. (B) Group data (mean±s.e.m) for EDD: end diastolic diameter (a); ESD: end systolic

diameter (b); FS: fractional shortening (c); PWd: posterior wall dimension (d); r/h: ratio of LV radius

to PWd (e); EF: ejection fraction (f); *p<0.01, **p<0.001 and ***p<0.0001 vs des-/-. (C) Survival

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curve of 3-month-old wt (n=12), des-/- (n=12) and des-/-αBCry (n=13) mice in a swimming protocol.

The error bars correspond to mean±s.e.m.; p<0.0001 des-/- vs wt and des-/-αBCry at all time points.

(D) Myocardial tissue ultra-structure from 3-months-old mice examined by transmission electron

microscopy; scale bar: 2 µm. (E) Higher magnification 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 bar: 0,2 µm.

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Fig. 4. αΒ-crystallin overexpression provides anti-oxidant protection to des-/- cardiomyocytes,

inhibits abnormal activation of the mPTP and the dissipation of Δψm. (A) Staining of live adult

cardiomyocytes in culture with CM-H2DCFDA to detect ROS, in control conditions and after

challenging with H2O2. Color graduation from light blue to brown corresponds to the intensity of CM-

H2DCFDA fluorescence and consequently the level of ROS; scale bar: 250 μm. (B) (a) Semi-

quantitative determination of CM-H2DCFDA intensity. Values are normalized to wt (mean±s.e.m.);

n=9; **p<0.01 vs wt, *p<0.05, vs des-/-. (b) Same as (a) after incubation with H2O2; ***p<0.001 and

#p≤0.05 vs wt, **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 wt heart while

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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 wt, (b) ***p≤0.001 vs wt, *p≤0.05 vs des-/-; n=4.

(D) Mitochondrial Δψ determination 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. (F) Mitochondrial Δψ loss analysis using 20 min time-lapse confocal

imaging of cardiomyocytes loaded with TMRM probe (see also Fig. S2). The mPTP inhibitor

Cyclosporin A was used to confirm the mPTP activation. The error bars show mean±s.e.m.; p<0.01 vs

des-/- and CsA; n=9.

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Fig. 5. Desmin and αB-crystallin bind to Mic60 and ATP synthase β. (A) WB analysis of total and

mitochondrial fractions isolated from hearts of the indicated genotypes. Mic60 is expressed as two

isoforms of 88 and 90 kDa. (B) Densitometric analysis of mitochondrial 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 wt, ^ p<0.001 vs wt. (C) GST pull-down assay using wt

mouse heart mitochondrial extract; arrowhead: nonspecific binding of 55 KDa IgGs. (D) Co-

immunoprecipitation analysis using cardiac mitochondrial extracts; arrowheads: nonspecific binding

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of 55 and 25 KDa IgGs. (E) ATP content of adult cardiomyocytes normalized to wt (mean±s.e.m,

n=8); ***p≤0.0001 vs wt, *p<0.05 vs des-/-.

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Fig. 6. Desmin and αB-crystallin are located at the SR and MAMs. (A) Cardiac mitochondria from

wt (a) and des-/-αBCry (b) mice were digested with proteinase K under the indicated conditions.

VDAC and mitofusin 2 were used as OMM markers, while ANT as IMM marker. (B) Subcellular

fractions from wt hearts were analyzed by WB for known markers of cytosol (a-actinin), mitochondria

(mit: MnSOD), MAMs and LM (light membranes mostly containing SR: VDAC, Mfn2, RYR). (C)

αB-crystallin subcellular localization in wt (1), des-/- (2) and des-/-αBCry (3) hearts. (D) Electron

micrographs of double immunoglod labeling of desmin (10 nm particle) and VDAC (25 nm particle).

Desmin is found at Z-discs (z, arrows), as expected (a), and in intimate association with mitochondria

(m, arrowheads) (a-c, a’-c’) and membrane structures attaching mitochondria, most possibly SR and

MAMs (white arrows) (c, c’), where it co-localizes with VDAC (white arrowheads) (a, a’, b, b’). a’-

c’ are higher magnifications of the boxed areas; scale bar 0.1 μm.

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Fig. 7. Desmin and αB-crystallin are found in the same supercomplex with Mic60 and VDAC.

GST pull-down assay (A) and VDAC immunoprecipitation analysis (B) using cardiac mitochondrial

lysates; arrowheads: nonspecific binding of 25 KDa IgGs. (C) Schematic representation of desmin (a),

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 a-helix N-terminus

of VDAC, Δβ1β4: deletion of the first 4 β-strands. (D) GST pull-down analysis of desmin and VDAC

domain interactions using mitochondrial lysates from wt and, in the case of VDAC, des-/- hearts. (E)

Heart mitochondria, solubilized with digitonin, were analyzed by blue native-PAGE in the first

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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 in the second

dimension by SDS-PAGE from animals in control conditions and after swimming exercise; red box:

macromolecular complexes of Mic60 diminished in the absence of desmin and restored by αB-

crystallin overexpression, blue box: shift of Mic60 and VDAC macromolecular complexes after

swimming stress.

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Tables

Table 1. αB-Crytallin overexpression restores several mitochondrial enzymes/proteins

diminished in the des-/- heart

Protein Name

Accession

Name

(MOUSE)

wt

vs

des-/-

des-/- αBCry

vs

des-/-

Alpha-crystallin B chain CRYAB - 66

Mic60 (Mitochondrial inner membrane protein) IMMT 4,46 3,6

Dihydrolipoyl dehydrogenase DLDH 2,13 3,7

3-mercaptopyruvate sulfurtransferase THTM 3,6

Pyruvate dehydrogenase E1 component subunit beta ODPB 0,43 3,4

2-oxoglutarate dehydrogenase ODO1 - 3,2

Succinyl-CoA ligase [ADP-forming] subunit beta SUCB1 - 3,5

Trifunctional enzyme subunit alpha ECHA 2,8 2,6

Succinate dehydrogenase [ubiquinone] flavoprotein

subunit DHSA 1,84 3,7

NADH dehydrogenase [ubiquinone] iron-sulfur

protein 2 NDUS2 1,44 2,3

NADH dehydrogenase [ubiquinone] 1-alpha

subcomplex subunit 10 NDUAA - 2

Electron transfer flavoprotein-ubiquinone

oxidoreductase ETFD 1,72 2,1

NADH dehydrogenase [ubiquinone] 1-beta

subcomplex subunit 9 NDUB9 1,98 -

NADH dehydrogenase [ubiquinone] flavoprotein 1 NDUV1 3,5 -

Some of the differentially expressed proteins in purified cardiac mitochondria extracts from 3-

months-old mice. Samples were analyzed by 2-dimensional gel electrophoresis coupled to mass

spectrometry (n=4) and differentially expressed protein spots were identified by MALDI-TOF/TOF-

MS. All the identified proteins are provided in Tables S3 and S4.

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Supplementary Information

Supplementary Figures

Fig. S1. A. Identification of αB-Crystallin transgenic founders by southern blot analysis.

From the lines generated two were chosen for further analysis, αBCry4 and αBCry6, with

high and low copy number of the transgene. A fragment of aMHC promoter was used as a

probe. B. Localization of αB-Crystallin at the intercalated discs. Double

immunofluorescence staining with αΒ-Crystallin and plakoglobin antibodies of frozen

myocardial tissue sections from non-stressed wild type (wt) and desmin null (des-/-) mice.

αB-Crystallin co-localizes with the desmosomal protein plakoglobin at the intercalated discs

(arrows) of both wt and desmin deficient hearts. Loss of αΒ-Crystallin from Z-discs in the

absence of desmin is also shown here. Scale bar: 10μm.

J. Cell Sci. 129: doi:10.1242/jcs.192203: Supplementary information

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Fig S2. Restoration of the diminished mitochondrial membrane potential of desmin

deficient cardiomyocytes by αB-Crystalin overexpression and/or Cyclosporin A. Time

lapse confocal imaging of adult cardiomyocytes loaded with the potentiometric indicator

TMRM with or without addition of the mitochondrial permeability transition pore inhibitor

Cyclosporin A. The florescence of TMRM was monitored for 20min with 1min interval; 5

different time points are shown in the figure; scale bar: 50µm.

J. Cell Sci. 129: doi:10.1242/jcs.192203: Supplementary information

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Fig. S3. i) Differentially expressed proteins in mitochondrial extracts isolated from heart

tissue of wt versus des-/- (A) and des-/-αBCry versus des-/- (B) mice. Representative 2D

gel images of wild type (wt), desmin null (des-/-) and desmin nul overexpressing αB-

Crystallin (des-/-αBCry) animals are shown. Differentially expressed spots (p<0.05, Mann-

J. Cell Sci. 129: doi:10.1242/jcs.192203: Supplementary information

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Whitney and Student’s t-test, 4 gels per category corresponding to 4 biological replicates) are

shown. In every case, 100 μg of total protein was analyzed in 7 cm non linear strips pH range

3-10 (BioRad). Gels were stained with coomassie colloidal blue and spot identification was

conducted by MALDI-TOF/TOF-MS (Bruker Daltonics). Protein identifications are provided

in supplementary tables S3 and S4. ii) Rescue of Mic60 containing macromolecular

complexes of des-/- mitochondria by αB-Crystallin overexpression. C) 50µg of solubilized

mitochondria from wt and wtαBCry mice were analyzed by 2-dimentional blue native/ SDS

PAGE and western blotted against VDAC, Mic60, αB-Crystallin and desmin antibodies.

Their localization in the same macromolecular complex is shown in the right panel. D) 60µg

of solubilized mitochondria from des-/- mice and 25µg from des-/-αBCry mice were analyzed

by 2D BN/SDS PAGE and probed against Mic60 and VDAC. Loading of lower amounts of

solubilized des-/-αBCry mitochondria relative to des-/-, strongly demonstrates the recovery of

larger (and smaller) Mic60 complexes. Red boxes indicated the macromolecular complexes

lost in mitochondria from desmin deficient mice.

J. Cell Sci. 129: doi:10.1242/jcs.192203: Supplementary information

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Fig. S4. Localization of desmin and VDAC by immunogold labeling of wt myocardial

ultra thin sections. Double labeling immunoelectron microscopy was performed using rabbit

polyclonal desmin antibody (decorated by 10 nm immunogold particles) and mouse

monoclonal VDAC antibody (decorated by 25 nm immunogold particles), as described in

Material and Methods. Desmin is detected at the expected structures, like intercalated disks

(IDs; arrows) (A) and the periphery of Z discs (Z), from which, occasionally, desmin positive

structures extend towards mitochondria (shown by arrowheads) (B). Furthermore, desmin is

also found in membranous structures adjacent to mitochondria, most likely sarcoplasmic

reticulum (SR) and MAMs (mitochondria-associated membranes) (arrows) (C) and in direct

J. Cell Sci. 129: doi:10.1242/jcs.192203: Supplementary information

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association with mitochondria where it co-localizes with VDAC (D). Boxes are higher

magnifications of the area. Scale bar: 0,1 µm. The experiments were performed with the

appropriate controls to evidence the specificity of immunogold labeling. In panel E anti-

desmin antibody was omitted, while the immunogold labeling procedure was executed as

usual using wt myocardial ultra thin sections. In panel F immunogold labeling with desmin

antibody was performed using des-/- myocardial ultra thin sections (scale bars: 1μm).The

VDAC monoclonal antibody labeling is not as efficient as a corresponding polyclonal

antibody, the use of which (decorated by 15 nm immunogold particles) in a single labeling

experiment, indeed, allows for a higher representation of the protein on mitochondria (G)

(scale bar: 0,1 μm). Decoration of desmin with nanogold particles, enhanced with silver (large

particles), and VDAC with 10 nm (small) immunogold particles, shows their close association

(H, I). Black arrow: desmin, white arrow: VDAC, scale bar: 0,2 µm. J) Quantification of

desmin and VDAC particles from several experiments (160 micrographs, 700 mitochondria),

revealed that desmin is found in 45,70% of the counted mitochondria, VDAC in 58,16%,

VDAC and desmin together in the same mitochondria (VDAC/desmin) in 29,82% and they

co-localize (VDAC+desmin) in 12,20% of them. Desmin is also found in mitochondria

associated membranes at 14,39% of the counted corresponding areas.

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Supplementary Tables

Table S1. Echocardiographic Parameters

wt des-/- des-/-αΒCry

EDD (mm) 3.50±0.47* 3.97±0.43 3.44±0.44*

ESD (mm) 1.92±0.30 *** 2.80±0. 36 2.00±0.34 ***

FS (%) 45.30±3.21 *** 29.62±2.79 41.83±5.43 ***

EF% 83.47±2.95*** 64.99±4.40 79.83±6.31***

PWT (mm) 0.87±0.10 ** 0.68±0.08 0.90±0.17 ***

r/h 2.04±0.32 *** 2.96±0.38 1.96±0.32 ***

n 11 14 11

Values are mean values ± s.d. WT: wild type, EDD: end diastolic diameter, ESD: end systolic

diameter, FS: Fractional Shortening, EF: ejection fraction PWT: posterior wall thickness, r/h:

ratio of LV radius to PWT. *

p<0.01 vs des-/- ; **

p<0.001 vs des-/-; *** p<0.0001 vs des-/-.

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Table S2. Desmin deficient mitochondria are susceptible to Ca2+

induced mPTP

activation and they can be protected by αB-Crystallin overexpression

OD540 decrease

control CaCl2 CaCl2 + CsA

wt 0.9583 ±0.0321 0.8334 ±0.0267** 0.9632±0.0480*

des-/- 0.9226 ±0.0253 0.7555±0.0222**# 0.9049±0.0055*

des-/- αBCry 0.9471±0.0210 0.8381±0.0296** 0.9584 ±0.0340*

wtαBCry 0.9444±0.0189 0.8347±0.0260** 0.9345±0.0390*

n 8 8 5

Overexpression of αB-Crystallin in des-/- heart improves the ability of des-/-mitochondria to

resist calcium and prevents abnormal mitochondrial PTP opening. Data are expressed as mean

percentage ± s.e.m. decrease in OD540 absorbance from time 0 to 30min. To each experiment

mitochondria isolated from one 2-month-old heart for each genotype were used. n represents

the number of experiments performed; ** p<0.01 CaCl2 vs control, * p<0.05 CaCl2 +CsA vs

CaCl2, # p< 0,05 des-/- vs the other genotypes. No statistical significant differences were

detected among genotypes at the control and CaCl2+CsA conditions. CsA: Cyclosporin A.

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Table S3. Differentially expressed proteins in cardiac mitochondrial extracts from

desmin deficient mice

Protein

Annotation

Number

Protein Name

Accession

Name

(Mouse)

wt/

des-/-

ratio

Mascot

Score

Sequence

Coverage

%

MW(kDa)

/pI

wt (density

in

ppm±SD)

des -/-

(density

in

ppm±SD)

1 Myosin light chain 3 MYL3 3.6 78 38 22.5/4.9 1122±278 314±55

2

ATP synthase subunit

beta, mitochondrial /

Prohibitin

ATPB /

PHB 0.33 127/103 42/57

56.3-

29.9/5.1-

5.47

404±67 1229±360

3

Pyruvate dehydrogenase

E1 component subunit

beta, mitochondrial

ODPB 0.43 124 63 23.2/4.5 3413±801 7963±169

7

4

NADH dehydrogenase

[ubiquinone] iron-sulfur

protein 2, mitochondrial

NDUS2 1.44 208 55 53/6.6 811±170 564±103

5

Dihydrolipoyllysine-

residue

succinyltransferase

component of 2-

oxoglutarate

dehydrogenase complex,

mitochondrial

ODO2 2.13 140 34 49.3/9.1 2308±180 1083±357

6

Dihydrolipoyllysine-

residue acetyltransferase

component of pyruvate

dehydrogenase complex,

mitochondrial

ODP2 1.53 154 35 68.5/9.6 3813±519 2490±724

7

Aldehyde dehydrogenase,

mitochondrial / ATP

synthase subunit beta,

mitochondrial

ALDH2 /

ATPB 1.48 208/63 53/34

57-

56.3/8.6-

5.07

1105±111 747±158

8

Succinate dehydrogenase

[ubiquinone] flavoprotein

subunit, mitochondrial

DHSA 1.84 256 62 73.6/7.3 1384±246 754±247

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9

Propionyl-CoA

carboxylase alpha chain,

mitochondrial / ATP

synthase subunit beta,

mitochondrial /

Trifunctional enzyme

subunit alpha,

mitochondrial

PCCA /

ATPB /

ECHA

2.8 251/64/

54 55/39/29

80.5-56.3-

83.3/6.9-

5.07-9.9

713±161 255±94

10 Voltage-dependent anion-

selective channel protein 2 VDAC2 0.46 98 50 32.3/8.7 443±146 968±48

11

Short-chain specific acyl-

CoA dehydrogenase,

mitochondrial

ACADS 0.2 191 59 45.1/9.4 297±91 1471±482

12

Succinate dehydrogenase

[ubiquinone] flavoprotein

subunit, mitochondrial

DHSA 0.23 67 25 73.6/7.3 262±62 1133±161

13

Dihydrolipoyl

dehydrogenase,

mitochondrial

DLDH 1.56 220 59 54.8/9.00 1334±280 858±109

14

Electron transfer

flavoprotein-ubiquinone

oxidoreductase,

mitochondrial

ETFD 1.72 214 43 68.9/7.9 995±169 578±116

15 Mitofilin (Mitochondrial

inner membrane protein) IMMT 4.46 336 51 84.2/6.2 3270±1229 733±174

16 Aconitate hydratase,

mitochondrial ACON 4.23 295 48 86.2/8.9 3481±967 824±254

17

NADH dehydrogenase

[ubiquinone] 1 beta

subcomplex subunit 9

NDUB9 1.98 119 67 22.3/8.8 5300±1028 2677±111

9

18

ES1 protein homolog,

mitochondrial /

Cytochrome b-c1 complex

subunit Rieske,

mitochondrial

ES1 /

UCRI 1.99 139/74 69/38

28.4-

29.6/9.9-

9.71

833±195 417±125

19

NADH dehydrogenase

[ubiquinone] 1 beta

subcomplex subunit 10

NDUBA 1.6 144 71 21.3/9.1 9324±1261 5815±192

7

20 Malate dehydrogenase,

mitochondrial MDHM 2.1 175 52 36/9.8 4296±1059 2082±582

21

Voltage-dependent anion-

selective channel protein

1/Hydroxyacyl-coenzyme

A dehydrogenase,

VDAC1 /

HCDH 0.34 146/66 66/33

30.9-

34.6/9.20-

9.46

3676±1859 10845±26

71

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mitochondrial

22

NADH dehydrogenase

[ubiquinone] flavoprotein

1, mitochondrial/ATP

synthase subunit alpha,

mitochondrial

NDUV1 /

ATPA 3.5 318/57 79/28

51.5-

59.8/9.5-

9.66

2494±535 708±213

23

EH domain-containing

protein 4/Hydroxysteroid

dehydrogenase-like

protein 2

EHD4 2.26 112 35 61,7 149±30 66±25

Differentially expressed proteins (p<0.05) were determined in mitochondrial extracts isolated

from mouse heart tissue of wild type versus desmin deficient animals following proteomics

and image analysis. Protein accession name (Swiss-Prot), identification parameters and fold

difference are provided. Location of proteins in the gel is shown in Supplemental Figure S3A.

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Table S4. αB-Crystallin overexpression restores the diminished levels of important mitochondrial proteins caused by desmin deficiency

Protein

Annotation

Number

Protein Name

Accession

Name

(MOUSE)

des-/-aBcry/

des-/-

ratio

Mascot

Score

Sequence

Coverage

%

MW(kDa)

/pI

des-/-aBcry

(density in

ppm±SD)

des-/-

(density in

ppm±SD)

1 Myosin light chain 3 MYL3 4.3 78 38 22.5/4.9 2967±1105 698±363

2 Alpha-crystallin B chain CRYAB 66 206 70 20.1/6.9 36521±1759

6 550±257

3

Dihydrolipoyl

dehydrogenase,

mitochondrial

DLDH 3.7 220 59 54.8/9.00 3141±922 842±202

4

Dihydrolipoyllysine-residue

succinyltransferase

component of 2-oxoglutarate

dehydrogenase complex,

mitochondrial

ODO2 2.1 153 36 49.3/9.1 2137±627 1027±341

5

3-mercaptopyruvate

sulfurtransferase / Citrate

lyase subunit beta-like

protein, mitochondrial

THTM /

CLYBL 3.6 116/73 47/37

33.2-

37.9/6.1-

9.58

727±196 201±69

6 Aldehyde dehydrogenase,

mitochondrial ALDH2 2.2 281 49 57/8.6 1512±461 678±286

7 Acyl-coenzyme A

thioesterase 2, mitochondrial ACOT2 1.8 174 49 49.9/7.1 1093±175 600±272

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8

Pyruvate dehydrogenase E1

component subunit beta,

mitochondrial

ODPB 3.4 144 59 39.3/6.5 637±185 187±85

9

NADH dehydrogenase

[ubiquinone] iron-sulfur

protein 2, mitochondrial

NDUS2 2.3 210 55 53/6.6 1650±554 705±189

10

Delta(3,5)-Delta(2,4)-

dienoyl-CoA isomerase,

mitochondrial

ECH1 2.1 191 55 36.4/8.8 9090±2550 4388±1595

11

NADH dehydrogenase

[ubiquinone] 1 alpha

subcomplex subunit 10,

mitochondrial

NDUAA 2 208 60 40.9/8.5 7141±1933 3516±1338

12

Succinate dehydrogenase

[ubiquinone] flavoprotein

subunit, mitochondrial

DHSA 3.7 373 69 73.6/7.3 4240±1796 1151±677

13

2-oxoglutarate

dehydrogenase,

mitochondrial

ODO1 3.5 234 33 117.6/6.4 2116±571 606±279

14

Short-chain specific acyl-

CoA dehydrogenase,

mitochondrial

ACADS 0.4 191 59 45.1/9.4 568±291 1422±481

15

Electron transfer

flavoprotein-ubiquinone

oxidoreductase,

mitochondrial

ETFD 2.1 129 30 68.9/7.9 924±255 450±178

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16 Mitofilin (Mitochondrial

inner membrane protein) IMMT 3.6 314 50 84.2/6.2 2535±1020 711±143

17 Aspartate aminotransferase,

mitochondrial AATM 2.3 291 66 47.8/9.8 15070±4208 6604±4168

18 Trifunctional enzyme subunit

alpha, mitochondrial ECHA 2.6 313 54 83.3/9.9 6620±2244 2562±1467

19

Succinyl-CoA ligase [ADP-

forming] subunit beta,

mitochondrial

SUCB1 3.7 205 50 50.4/6.7 4537±1868 1241±475

Differentially expressed proteins (p<0.05) in mitochondrial extracts isolated from mouse heart tissue of desmin deficient overexpressing αB-Crystallin (des-/-

αBCry) versus desmin deficient (des-/-) animals following proteomics and image analysis. Protein accession name (Swiss-Prot), identification parameters and

fold difference are provided. Location of proteins in the gel is shown in Supplemental Figure S3B.

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