Post on 20-May-2020
© 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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