BRFAA - Biomedical Research Foundation Academy Of Athens
Biomedical Research Foundation Academy Of AthensAcademy Of Athens


Unravelling the mechanisms responsible for heart failure development and strategies for its prevention.

Heart failure is a world-wide public leading cause of death. The molecular and cellular mechanisms underlying heart failure remain elusive. Cardiomyocyte death is considered a major cause in the development of dilated cardomyopathy and heart failure, possibly due to disturbance of the cardiomyocyte cytoskeletal network and calcium homeostasis. Disturbance in several members of this network leads to the same disease, that is dilated cardiomyopathy and heart failure. A major part of Capetanaki’s research has focused on the elucidation of the mechanism by which perturbations in the cytoskeleton, and most specifically deficiency, mutations or caspase cleavage of the muscle specific intermediate filament protein desmin leads to mitochondrial defects and cardiomyocyte death. For this purpose, we follow two strategies: One will address the cardiomyocyte intrinsic molecular and cellular mechanisms leading to mitochondrial defects, cell death and heart failure and the second will elucidate the contribution of extrinsic factors (including inflammation) in the progress of the disease. These efforts have already opened very promising paths towards not only our better understanding of the function of desmin in cardiomyopathy but furthermore, the development of strategies to reverse this process.

I. Mechanisms Responsible for Heart Failure Development

Study on the cardiomyocyte-intrinsic molecular and cellular mechanisms
Our studies on desmin null mice have demonstrated that desmin is very important in muscle maintenance and its absence leads to cardiomyopathy and heart failure. The hallmarks of the cardiomyopathy are mitochondrial defects, muscle degeneration with extensive fibrosis and calcification. The mechanism by which desmin intermediate filaments (IFs) contribute to maintenance of healthy muscle and the mechanism by which lack of functional desmin IFs leads to myocyte death remains elusive. It is our goal to address this issue. Our studies so far have established that the presumed association of desmin IFs with mitochondria is of great significance for the maintenance of healthy heart. Among the most prominent and early detected features of the desmin null phenotype are defects on mitochondrial shape, morphology, structure and positioning, compromised respiratory function and mitochondria proliferation and swelling, especially following work overload. There are different ways by which the observed mitochondrial abnormalities could lead to cell death. These include: a) impaired mitochondrial function; b) increase in oxidative stress; c) perturbation of calcium homeostasis; d) changes in mitochondrial membrane permeability; and e) release of cell death molecules. We are addressing each one of these possibilities and try to establish the potential overlap between them. Towards the above goals we have either been using as a model our desmin null mice or we have generated and are characterizing mice with desmin point mutations found in human patients with heart failure.

α.  Ιmpaired mitochondrial function
We have already established that desmin deficiency leads to respiratory dysfunction and have indications of increase in oxidative stress and perturbation of calcium homeostasis. In an effort to unravel the mode and the mechanism responsible for these deficiencies we investigated potential mitochondrial composition changes using proteomic analysis. We have observed differences in most metabolic pathways, in apoptosis, calcium homeostasis, calcification and fibrosis and in different signaling pathways linked or not to mitochondrial function. The most significant changes were observed in ketone body and acetate metabolism, NADH shuttle proteins, amino acid metabolism proteins and respiratory enzymes. Several of these changes are consistent with the known phenotype of desmin deficiency. Most importantly changes in proteins of the MPTP super channel are of great importance and are further investigated (J. Mol. Cell. Cardiol. 2005).

b.  Increase in oxidative stress (Diokmentzidou)
We have demonstrated increased oxidative stress in the desmin deficient cardiomyocytes. In an effort to establish the observed oxidative stress as a cause of cell death and myocardial degeneration, we have generated transgenic mice overexpressing antioxidant enzymes, such as MnSOD and catalase, in the heart and study the consequences in cardioprotection. Moderate overexpression of catalase in desmin null hearts leads to a marked decrease in intracellular reactive oxygen species (ROS) levels, ameliorates myocardial degeneration and leads to a significant decrease in fibrotic areas and improvement of heart function (Rapri et al., in preparation). Our long term goal is to use the obtained information from these functional studies to develop therapeutic approaches for cardiomyopathies using the desmin deficient mouse models.

c.  Changes in mitochondrial membrane permeability
Several of our studies suggested that an important part of the mitochondria that might be affected in desmin deficient cardiomyocytes might be the contact sites and important structures located there including components that control mitochondria membrane permeability. This led to the hypothesis that important factors controlling this permeability, such as the anti-apoptotic protein Bcl2, might be compromised and increase in its expression might help. Indeed this hypothesis was correct and the designed experiments allowed the outcome of one of the major contributions of the laboratory. That was our success to rescue heart failure in desmin null mice by over-expressing bcl-2 in the heart (PNAS 2004). This is of great significance because it shows for the first time that bcl-2, a mitochondrial antiapoptotic protein, can provide significant relief to an inherited form of dilated cardiomyopathy (DCM) caused by perturbation of the cytoskeleton and thus suggesting its potential use for intervention in DCM generally, given the fact that mutations in different cytoskeletal proteins lead to this cardiomyopathy.

αB-Crystallin Overexpression Mediates Protection of the Desmin Null Myocardium (Diokmetzidou)

We have also tested the proposed hypothesis that desmin is essential for effective cardioprotective function of some desmin associated proteins, such as the small stress or heat shock proteins (shsps). Transgenic mice overexpressing αB-crystallin in the heart have been generated. αB-Crystallin overexpression provides significant improvement of cardiac morphology and histological appearance as demonstrated by decreased fibrosis and calcification and preservation of tissue strength and integrity. Ultrastructural defects of the desmin null heart, such as mitochondrial swelling and destruction and myofibril disarray have been corrected to a significant degree as evidenced by transmission electron microscopy. Cardiac function was ameliorated to a level that is similar to that of wild type, as shown by improvement of systolic function, increase in posterior wall thickness and reduction of LV wall stress. In addition, in the case of αΒ-Crystallin overexpression, desmin null mice demonstrate a rate of 100% survival in an obligatory exercise swimming protocol, while in the same conditions, only 50% of desmin null mice survive. Also, αΒ-Crystallin overexpression provides significant protection against endogenous and exogenous oxidative stress, Moreover, αΒ -crystallin protects against the abnormal activation of mPTP and the decrease of mitochondrial potential (Δψ). We believe that αΒ -Crystallin may provide protection to the desmin null myocardium by acting as a pleiotropic inhibitor of cell death (Diokmetzidou, Soumaka et al., EHJ, under revision). These studies are extended to other heart failure models, to establish its effectiveness in heart failure, in general, regardless of the cause of origin.

Desmin as a major mediator of heart failure

Desmin is a target and mediator of TNF-α induced caspase-cleavage , aggregate formation and intercalated disk reorganization

We explored the involvement of the muscle specific intermediate filament protein desmin in the model of TNF- induced cardiomyopathy. We demonstrate that in mice overexpressing TNF-α in the heart (MHCsTNF), desmin is modified, loses its intercalated disk (ID) localization, and forms aggregates that colocalize with HSP25 and ubiquitin. Additionally, other ID proteins, such as desmoplakin and β-catenin, show similar localization changes in a desmin-dependent fashion. To address underlying mechanisms we examined whether desmin is a substrate for caspase-6 in vivo, as well as the implications of desmin cleavage in MHCsTNF mice. We generated transgenic mice with cardiac-restricted expression of a desmin mutant (D263E), and proved that it is resistant to caspase cleavage in the MHCsTNF myocardium. The aggregates are diminished in these mice, and D263E desmin, desmoplakin and β-catenin largely retain their proper ID localization. Importantly, D263E desmin expression attenuated cardiomyocyte apoptosis, prevented left ventricular wall thinning and improved function of MHCsTNF hearts (Panagopoulou et al., J.Cell Biol. 2008).

Cardioprotection by TNF-α overexpression in desmin deficient heart failure (Papathanasiou)

In an effort to further determine that the TNF-α - induced abnormalities were indeed desmin dependent, desmin null mice overexpressing TNF-α in the heart (TNF-α des-/-) were generated. To our surprise, we found that TNF-α over-expression in the absence of desmin, has an impressive cardioprotective effect, manifested by the absence of the desmin null heart pathology. Despite the presence of interstitial fibrosis and hypertrophy, basic characteristics of the TNF-α des+/+ myocardium, the TNF-α des-/- animals exhibit improved cardiac function and viability, compared to the former ones. To further elucidate the mechanisms by which TNF-α overexpression ameliorates most of the defects developed in desmin deficient hearts, gene expression profile analysis has been performed, resulting in a list of several interesting genes that are currently being studied.

Identification of novel desmin-interacting proteins

In an effort to unravel the function of the desmin tail and head domains yeast two hybrid system strategies have been employed for the identification of novel proteins interacting with desmin. The initial results are very exciting and if these interactions will be confirmed also by other methods, novel functions of desmin will be established and new areas of investigations of their mechanisms will be initiated.

Determination of the role of the desmin associated protein Myospryn in vitro and in vivo (Tsoupri)

The desmin amino-terminal domain binds the TRIM-like Protein Myospryn

Desmin surrounds the Z-discs and links the entire contractile apparatus to the sarcolemmal cytoskeleton, cytoplasmic organelles and the nucleus. In an attempt to explore the molecular mechanisms of these associations we performed a yeast two-hybrid screening of a cardiac cDNA library. We showed that the desmin amino-terminal domain [Naa(1 -103)] binds to a 413kDa TRIM-like protein, myospryn, which is expressed in cardiac and skeletal muscle. Myospryn was found to interact with dysbindin, a component of the Biogenesis of Lysosome Related Organelles Complex 1 (BLOC-1), which is involved in protein trafficking and organelle biogenesis. Binding of desmin with myospryn was confirmed in vitro with GST pull down assay and co-immunoprecipitation experiments. Deletion analysis revealed that only the [Naa(1 -103)] fragment of desmin binds to myospryn carboxyl terminus and that this association takes place through the 24aa long C-terminal end of the SPRY domain of myospryn. Using an antibody against the COOH terminus of myospryn, we demonstrated that myospryn co-localizes with desmin at the periphery of the nucleus of mouse neonatal cardiomyocytes. In adult heart muscle, the two proteins colocalize, predominantly, at intercalated discs and costameres. We also showed that myospryn colocalizes with lysosomes as well. Furthermore we determined that desmin is required for both the proper perinucler localization of myospryn, as well as the proper positioning of lysosomes, thus suggesting a potential role of desmin IFs in lysosomes and lysosome-related organelle biogenesis and/or positioning (Kouloumenta et al., J. Biol. Chem. 2007).
Using siRNA and mouse two-hit conditional tet-off system knock-in strategies, we are trying to determine the importance of myospryn-desmin interaction in signaling as well as in trafficking and organelle biogenesis, in tissue culture and in vivo.

Unravelling the role of desmin in mechanotransduction and its implications in heart disease

Using both antisense strategies and knock out experiments in embryonic stem cells, our work has previously suggested for the first time that desmin intermediate filaments might participate in mechanochemical signaling from the Z- line of the contractile apparatus to the nucleus since its inhibition interfered with the expression of myogenic transcription regulators (J. Cell Biol. 1994; Dev. Biol. 1995). Mechanotransduction plays a fundamental role in the myocardium where its cycle of contraction and relaxation leads to dynamic deformations. The mechanism by which myocytes sense mechanical forces is still unknown. The hypothesis is that the Z-disc associated proteins could serve as mechanical stretch sensors and desmin, which surrounds the z-disc and links them to the nucleus, is a potential candidate for sensinig and transmitting the signals to the nucleus. This project will test this hypothesis. Towards this end, with the help of our engineers collaborators at San Diego we have directly shown using muscle deformation that desmin is linked to the nucleus and influences its shape and potentially gene activity (Shah et al., 2004). To further unravel the molecular mechanisms of these linkages to nucleus and Z discs we will focus on some known candidate molecules and other novel ones that have emerged from yeast-2 hybrid screens as well as RNA profiling.

Study of the cardiomyocyte extrinsic molecular and cellular mechanisms in the progress of heart failure

Regulation of Adverse Remodeling by Osteopontin in the des-/- Heart Failure Model

We have shown that myocardial degeneration in desmin deficient hearts is accompanied by inflammation, extended fibrosis, calcification , developing overtime DCM and heart failure. Therefore, in parallel to the study of the intrinsic cardiomyocyte defects leading to cell death, we wanted to identify the molecular and cellular events regulating the observed adverse cardiac remodeling in desmin deficient hearts. To address this issue we did initially a small scale DNA array study. The most substantial changes were found for genes coding for extracellular matrix proteins and especially for the small matricellular protein osteopontin (Mavroidis et al., AJP 2002). Osteopontin is a matricellular protein and a cytokine induced upon tissue injury and remodeling of various organs, including human failing heart, atherosclerosis, vascular remodeling and restenosis after angioplasty. Our hypothesis was that :1) the upregulation of osteopontin contributes to the progression of fibrosis and calcification, the pathology observed in desmin deficient cardiomyopathy, as well as in age- and diabetes-related dystrophic calcification in the human cardiovascular system;. and 2) targeted down regulation of osteopontin expression will decrease the extent of pathology. To test the above hypotheses we generated desmin null mice deficient in osteopondin and we have studied the effect of the absence of osteopondin in the progress of the myocardial pathology and cardiac function. We have further investigated the molecular and cellular events regulating adverse cardiac remodeling in des-/- mice by performing more extended whole genome RNA profile and In situ hybridization, histology and immunostaining demonstrated that inflammatory cells and not cardiomyocytes were the source of OPN. RNA profile comparison revealed that activation of inflammatory pathways predominated among all changes occurring in degenerating des-/- myocardium. The expression of the most highly up-regulated genes (OPN: 226x, galectin-3: 26x, osteoactivin/Gpnmb/DC-HIL: 160x and metalloprotease-12: 98x) was associated with heart infiltrating macrophages. To evaluate the role of OPN we generated des-/-OPN-/- mice and compared their cardiac function and remodeling indices with those of des-/-. OPN promoted cardiac dysfunction in this model since des-/-OPN-/- mice showed 53% improvement of left ventricular function, paralleled to an up to 44% reduction of fibrosis, as compared to des-/- counterparts. The diminished fibrotic response in the absence of OPN could be partly mediated by a dramatic reduction of myocardial galectin-3 levels, associated with an impaired galectin-3 secretion by OPN-deficient infiltrating macrophages. We have concluded Cardiomyocyte death due to desmin deficiency leads to inflammation and subsequent overexpression of a series of remodeling modulators. Among them, OPN seems to be a major regulator of des-/- adverse myocardial remodeling and it functions at least by potentiating galectin-3 up-regulation and secretion (Psarras et al. Eur. Heart J. 2011)


II. Development of novel therapeutic strategies for cardiomyopathies (Tsikiti, Nikouli, Diokmetzidou)

The other major goal of our group is to use the desmin deficient, mutant and TNF-α heart failure mouse models to develop A) efficient drug and gene-based therapeutic strategies targeting rescue of normal cardiomyocyte mitochondrial structure and function; B) cell –based strategies for myocardial regeneration.

A.  In pre-clinical translational studies, described above, we have demonstrated the cardioprotective role of the anti-apoptotic protein bcl2, small heat shock proteins (particularly aB-crystallin) and anti-oxidant enzymes in the desmin deficient heart failure model. We will continue and extend these studies with additional cardiomyopathy models.

B.   We have found that desmin is one of the earliest myogenic markers both in heart and in somites (Kuisk et al., 1996) as well as in embryoid body differentiation in vitro (Weitzer et al., 1995). Desmin is expressed in satellite cells, the skeletal muscle stem cells, as well as in the side population of the adult cardiac progenitor cells (CSP) and has been found to promote cardiogenesis in embryonic stem cells (ESCs) (Hollrigl et al., 2007). We have hypothesized that in addition to differentiation, desmin modulates cardiac stem cell maintenance and homing characteristics and we are testing this hypothesis. Specifically, we investigate the role of desmin in maintenance, differentiation and homing/transplantation efficiency of cardiac stem cells and in addition we are using the desmin-null cardiomyopathy model as a paradigm to optimize stem and non stem cell properties for heart repair.