Desmin deficiency affects the microenvironment of the cardiac side population and Sca1+ stem cell population of the adult heart and impairs their cardiomyogenic commitment.

The heart's limited regenerative capacity raises the need for novel stem cell-based therapeutic approaches for cardiac regeneration. However, the use of stem cells is restrictive due to poor determination of their properties and the factors that regulate them. Here, we investigated the role of desmin, the major muscle-specific intermediate filament protein, in the characteristics and differentiation capacity of cardiac side population (CSP) and Sca1+ stem cells of adult mice. We found that desmin deficiency affects the microenvironment of the cells and leads to increased numbers of CSP but not Sca1+ cells; CSP subpopulation composition is altered, the expression of the senescence marker p16INK4a in Sca1+ cells is increased, and early cardiomyogenic commitment is impaired. Specifically, we found that mRNA levels of the cardiac transcription factors Mef2c and Nkx2.5 were significantly reduced in des-/- CSP and Sca1+ cells, while differentiation of CSP and Sca1+ cells demonstrated that in the absence of desmin, the levels of Nkx2.5, Mef2c, Tnnt2, Hey2, and Myh6 mRNA are differentially affected. Thus, desmin deficiency restricts the regenerative potential of CSP and Sca1+ cells, both directly and indirectly through their microenvironment.

Galectin-3 interferes with tissue repair and promotes cardiac dysfunction and comorbidities in a genetic heart failure model.

Galectin-3, a biomarker for heart failure (HF), has been associated with myocardial fibrosis. However, its causal involvement in HF pathogenesis has been questioned in certain models of cardiac injury-induced HF. To address this, we used desmin-deficient mice (des-/-), a model of progressive HF characterized by cardiomyocyte death, spontaneous inflammatory responses sustaining fibrosis, and galectin-3 overexpression. Genetic ablation or pharmacological inhibition of galectin-3 led to improvement of cardiac function and adverse remodeling features including fibrosis. Over the course of development of des-/- cardiomyopathy, monitored for a period of 12 months, galectin-3 deficiency specifically ameliorated the decline in systolic function accompanying the acute inflammatory phase (4-week-old mice), whereas a more pronounced protective effect was observed in older mice, including the preservation of diastolic function. Interestingly, the cardiac repair activities during the early inflammatory phase were restored under galectin-3 deficiency by increasing the proliferation potential and decreasing apoptosis of fibroblasts, while galectin-3 absence modulated macrophage-fibroblast coupled functions and suppressed both pro-fibrotic activation of cardiac fibroblasts and pro-fibrotic gene expression in the des-/- heart. In addition, galectin-3 also affected the emphysema-like comorbid pathology observed in the des-/- mice, as its absence partially normalized lung compliance. Collectively galectin-3 was found to be causally involved in cardiac adverse remodeling, inflammation, and failure by affecting functions of cardiac fibroblasts and macrophages. In concordance with this role, the effectiveness of pharmacological inhibition in ameliorating cardiac pathology features establishes galectin-3 as a valid intervention target for HF, with additive benefits for treatment of associated comorbidities, such as pulmonary defects. Schematic illustrating top to bottom, the detrimental role of galectin-3 (Gal3) in heart failure progression: desmin deficiency-associated spontaneous myocardial inflammation accompanying cardiac cell death (reddish dashed border) is characterized by infiltration of macrophages (round cells) and up-regulation of Lgals3 (encoding secretable galectin-3, green) and detrimental macrophage-related genes (Ccr2 and Arg1). In this galectin-3-enriched milieu, the early up-regulation of profibrotic gene expression (Tgfb1, Acta2, Col1a1), in parallel to the suppression of proliferative activities and a potential of senescence induction by cardiac fibroblasts (spindle-like cells), collectively promote des-/- cardiac fibrosis and dysfunction establishing heart failure (left panel). Additionally, galectin-3+ macrophage-enrichment accompanies the development of emphysema-like lung comorbidities. In the absence of galectin-3 (right panel), the effect of macrophage-fibroblast dipole and associated events are modulated (grey color depicts reduced expression or activities) leading to attenuated cardiac pathology in the des-/-Lgals3-/- mice. Pulmonary comorbidities are also limited.

Myospryn deficiency leads to impaired cardiac structure and function and schizophrenia-associated symptoms.

The desmin-associated protein myospryn, encoded by the cardiomyopathy-associated gene 5 (CMYA5), is a TRIM-like protein associated to the BLOC-1 (Biogenesis of Lysosomes Related Organelles Complex 1) protein dysbindin. Human myospryn mutations are linked to both cardiomyopathy and schizophrenia; however, there is no evidence of a direct causative link of myospryn to these diseases. Therefore, we sought to unveil the role of myospryn in heart and brain. We have genetically inactivated the myospryn gene by homologous recombination and demonstrated that myospryn null hearts have dilated phenotype and compromised cardiac function. Ultrastructural analyses revealed that the sarcomere organization is not obviously affected; however, intercalated disk (ID) integrity is impaired, along with mislocalization of ID and sarcoplasmic reticulum (SR) protein components. Importantly, cardiac and skeletal muscles of myospryn null mice have severe mitochondrial defects with abnormal internal vacuoles and extensive cristolysis. In addition, swollen SR and T-tubules often accompany the mitochondrial defects, strongly implying a potential link of myospryn together with desmin to SR- mitochondrial physical and functional cross-talk. Furthermore, given the reported link of human myospryn mutations to schizophrenia, we performed behavioral studies, which demonstrated that myospryn-deficient male mice display disrupted startle reactivity and prepulse inhibition, asocial behavior, decreased exploratory behavior, and anhedonia. Brain neurochemical and ultrastructural analyses revealed prefrontal-striatal monoaminergic neurotransmitter defects and ultrastructural degenerative aberrations in cerebellar cytoarchitecture, respectively, in myospryn-deficient mice. In conclusion, myospryn is essential for both cardiac and brain structure and function and its deficiency leads to cardiomyopathy and schizophrenia-associated symptoms.

Skeletal and Cardiac Muscle Disorders Caused by Mutations in Genes Encoding Intermediate Filament Proteins.

Intermediate filaments are major components of the cytoskeleton. Desmin and synemin, cytoplasmic intermediate filament proteins and A-type lamins, nuclear intermediate filament proteins, play key roles in skeletal and cardiac muscle. Desmin, encoded by the DES gene (OMIM *125660) and A-type lamins by the LMNA gene (OMIM *150330), have been involved in striated muscle disorders. Diseases include desmin-related myopathy and cardiomyopathy (desminopathy), which can be manifested with dilated, restrictive, hypertrophic, arrhythmogenic, or even left ventricular non-compaction cardiomyopathy, Emery-Dreifuss Muscular Dystrophy (EDMD2 and EDMD3, due to LMNA mutations), LMNA-related congenital Muscular Dystrophy (L-CMD) and LMNA-linked dilated cardiomyopathy with conduction system defects (CMD1A). Recently, mutations in synemin (SYNM gene, OMIM *606087) have been linked to cardiomyopathy. This review will summarize clinical and molecular aspects of desmin-, lamin- and synemin-related striated muscle disorders with focus on LMNA and DES-associated clinical entities and will suggest pathogenetic hypotheses based on the interplay of desmin and lamin A/C. In healthy muscle, such interplay is responsible for the involvement of this network in mechanosignaling, nuclear positioning and mitochondrial homeostasis, while in disease it is disturbed, leading to myocyte death and activation of inflammation and the associated secretome alterations.

Three in a Box: Understanding Cardiomyocyte, Fibroblast, and Innate Immune Cell Interactions to Orchestrate Cardiac Repair Processes.

Following an insult by both intrinsic and extrinsic pathways, complex cellular, and molecular interactions determine a successful recovery or inadequate repair of damaged tissue. The efficiency of this process is particularly important in the heart, an organ characterized by very limited regenerative and repair capacity in higher adult vertebrates. Cardiac insult is characteristically associated with fibrosis and heart failure, as a result of cardiomyocyte death, myocardial degeneration, and adverse remodeling. Recent evidence implies that resident non-cardiomyocytes, fibroblasts but also macrophages -pillars of the innate immunity- form part of the inflammatory response and decisively affect the repair process following a cardiac insult. Multiple studies in model organisms (mouse, zebrafish) of various developmental stages (adult and neonatal) combined with genetically engineered cell plasticity and differentiation intervention protocols -mainly targeting cardiac fibroblasts or progenitor cells-reveal particular roles of resident and recruited innate immune cells and their secretome in the coordination of cardiac repair. The interplay of innate immune cells with cardiac fibroblasts and cardiomyocytes is emerging as a crucial platform to help our understanding and, importantly, to allow the development of effective interventions sufficient to minimize cardiac damage and dysfunction after injury.

Desmin deficiency is not sufficient to prevent corneal fibrosis.

The type III intermediate filament (IF) proteins vimentin and desmin are sequentially overexpressed in stromal myofibroblasts over the period when fibrosis sets in after corneal injury. Prior findings have revealed vimentin-deficient mice are significantly protected from corneal fibrosis after alkali injury, which has implicated this IF protein as an important regulator of corneal fibrosis. It has remained as yet unproven whether desmin contributes in any significant manner to corneal fibrosis. Here we have employed desmin-deficient (Des KO) mice in the corneal alkali injury model and show that injured Des KO mice develop fibrosis and show similar levels of corneal opacity at 14 days post-injury as wild type (WT) mice and retain this phenotype even at 30d post injury. Des KO corneas from injured mice show upregulation of vimentin and alpha-smooth muscle actin expression to equivalent levels as WT corneas, illuminating that desmin deficiency does not interfere with myofibrobast differentiation. Employing the small molecule withaferin A (WFA), an inhibitor of vimentin, we show that WFA treatment causes the decrease in steady state levels of vimentin and serine 38 phosphorylated vimentin, the latter a biomarker associated with corneal fibrosis, and improved corneal clarity through blockade of myofibroblast differentiation. To investigate further the mechanism of fibrosis in desmin deficiency, we examined keratin 8 expression in the epithelium, and found reduced levels of this cytokeratin in injured Des KO corneas compared to WT corneas. This finding also corroborates the decrease of cell proliferation in injured Des KO corneas compared to that in WT corneas. The fibrotic phenotype of Des KO corneas also features abundant vascularization, further exemplifying the magnitude of corneal pathology. Together, these findings illuminate that desmin does not contribute significantly to corneal fibrosis in this injury model.

Amelioration of desmin network defects by αB-crystallin overexpression confers cardioprotection in a mouse model of dilated cardiomyopathy caused by LMNA gene mutation.

The link between the cytoplasmic desmin intermediate filaments and those of nuclear lamins serves as a major integrator point for the intracellular communication between the nucleus and the cytoplasm in cardiac muscle. We investigated the involvement of desmin in the cardiomyopathy caused by the lamin A/C gene mutation using the LmnaH222P/H222P mouse model of the disease. We demonstrate that in these mouse hearts desmin loses its normal Z disk and intercalated disc localization and presents aggregate formation along with mislocalization of basic intercalated disc protein components, as well as severe structural abnormalities of the intercalated discs and mitochondria. To address the extent by which the observed desmin network defects contribute to the progression of LmnaH222P/H222P cardiomyopathy, we investigated the consequences of desmin-targeted approaches for the disease treatment. We showed that cardiac-specific overexpression of the small heat shock protein αΒ-Crystallin confers cardioprotection in LmnaH222P/H222P mice by ameliorating desmin network defects and by attenuating the desmin-dependent mislocalization of basic intercalated disc protein components. In addition, αΒ-Crystallin overexpression rescues the intercalated disc, mitochondrial and nuclear defects of LmnaH222P/H222P hearts, as well as the abnormal activation of ERK1/2. Consistent with that, by generating the LmnaH222P/H222PDes+/- mice, we showed that the genetically decreased endogenous desmin levels have cardioprotective effects in LmnaH222P/H222P hearts since less desmin is available to form dysfunctional aggregates. In conclusion, our results demonstrate that desmin network disruption, disorganization of intercalated discs and mitochondrial defects are a major mechanism contributing to the progression of this LMNA cardiomyopathy and can be ameliorated by αΒ-Crystallin overexpression.

Intermediate filaments in cardiomyopathy.

Intermediate filament (IF) proteins are critical regulators in health and disease. The discovery of hundreds of mutations in IF genes and posttranslational modifications has been linked to a plethora of human diseases, including, among others, cardiomyopathies, muscular dystrophies, progeria, blistering diseases of the epidermis, and neurodegenerative diseases. The major IF proteins that have been linked to cardiomyopathies and heart failure are the muscle-specific cytoskeletal IF protein desmin and the nuclear IF protein lamin, as a subgroup of the known desminopathies and laminopathies, respectively. The studies so far, both with healthy and diseased heart, have demonstrated the importance of these IF protein networks in intracellular and intercellular integration of structure and function, mechanotransduction and gene activation, cardiomyocyte differentiation and survival, mitochondrial homeostasis, and regulation of metabolism. The high coordination of all these processes is obviously of great importance for the maintenance of proper, life-lasting, and continuous contraction of this highly organized cardiac striated muscle and consequently a healthy heart. In this review, we will cover most known information on the role of IFs in the above processes and how their deficiency or disruption leads to cardiomyopathy and heart failure.

Type III Intermediate Filaments Desmin, Glial Fibrillary Acidic Protein (GFAP), Vimentin, and Peripherin.

SummaryType III intermediate filament (IF) proteins assemble into cytoplasmic homopolymeric and heteropolymeric filaments with other type III and some type IV IFs. These highly dynamic structures form an integral component of the cytoskeleton of muscle, brain, and mesenchymal cells. Here, we review the current ideas on the role of type III IFs in health and disease. It turns out that they not only offer resilience to mechanical strains, but, most importantly, they facilitate very efficiently the integration of cell structure and function, thus providing the necessary scaffolds for optimal cellular responses upon biochemical stresses and protecting against cell death, disease, and aging.

Opposite effects of catalase and MnSOD ectopic expression on stress induced defects and mortality in the desmin deficient cardiomyopathy model.

Oxidative stress has been linked strongly to cell death and cardiac remodeling processes, all hallmarks of heart failure. Mice deficient for desmin (des-/-), the major muscle specific intermediate filament protein, develop dilated cardiomyopathy and heart failure characterized by mitochondrial defects and cardiomyocyte death. The cellular and biochemical alterations in the hearts of these mice strongly suggest that oxidative stress is one of the mechanisms contributing to the pathogenesis of the phenotype. Recently, we showed that indeed the desmin deficient cardiomyocytes are under increased oxidative stress. In order to verify these findings in vivo, we generated transgenic animals overexpressing SOD2 (MnSOD) and/or catalase in the heart and crossed them with des-/- mice, thus allowing us to evaluate the contribution of oxidative injury in inherited cardiomyopathies, as well as the therapeutic potential of antioxidant strategies. Moderate MnSOD and/or catalase overexpression in des-/- hearts leads to a marked decrease in intracellular reactive oxygen species (ROS), ameliorates mitochondrial and other ultrastructural defects, minimizes myocardial degeneration and leads to a significant improvement of cardiac function. Importantly, catalase overexpression increased the 50% survival rate of des-/- mice in an obligatory exercise to 100%. In contrast, MnSOD overexpression enhanced the lethality of des-/- mice, underscoring the importance of a fine balanced cellular redox status. Overall, the present study supports the contribution of oxidative stress in the development of des-/- cardiomyopathy and points to a well-considered antioxidant treatment as therapeutic for cardiomyopathies.

Nebulette is a powerful cytolinker organizing desmin and actin in mouse hearts.

In the hearts of patients bearing nebulette mutations, a severe general disorganization in cardiomyocytes of the extrasarcomeric desmin intermediate filament system is frequently observed. However, the molecular and functional relationship between the desmin cytoskeleton and nebulette-containing sarcomeres is still unclear. Here we report a high-affinity in vitro interaction between nebulette and desmin filaments. A major interaction site has been mapped to the desmin α-helical rod domain, indicating that the filament core is directly involved in the binding of nebulette. The disease-mutant desmin variants E245D and T453I exhibited increased binding affinity for nebulette, delayed filament assembly kinetics, and caused significant weakening of networks. In isolated chick cardiomyocytes and sections from canine heart, we revealed by ground-state depletion and confocal microscopies that module 5 of nebulette extends outward from Z-disk-associated desmin filaments toward the center of the sarcomere. Accordingly, in the myocardium of Des-/- mice, elevated levels of cardiac actin correlated with alterations in the distribution of nebulette. Our data suggest that a well-organized desmin network is required to accommodate an optimal conformation of nebulette on sarcomeres to bind and recruit cardiac α-actin. Hence we propose that nebulette acts in synergy with nebulin to reinforce and temporally fine-tune striated muscle relaxation-contraction cycles.

Desmin and αB-crystallin interplay in the maintenance of mitochondrial homeostasis and cardiomyocyte survival.

The association of desmin with the α-crystallin Β-chain (αΒ-crystallin; encoded by CRYAB), and the fact that mutations in either one of them leads to heart failure in humans and mice, suggests a potential compensatory interplay between the two 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 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 (Δψm). Furthermore, we found that both desmin and αΒ-crystallin are localized at sarcoplasmic reticulum (SR)-mitochondria-associated membranes (MAMs), where they interact with VDAC, Mic60 - the core component of mitochondrial contact site and cristae organizing system (MICOS) complex - and ATP synthase, suggesting that these associations could be crucial in mitoprotection at different levels.

Desmin enters the nucleus of cardiac stem cells and modulates Nkx2.5 expression by participating in transcription factor complexes that interact with the nkx2.5 gene.

The transcription factor Nkx2.5 and the intermediate filament protein desmin are simultaneously expressed in cardiac progenitor cells during commitment of primitive mesoderm to the cardiomyogenic lineage. Up-regulation of Nkx2.5 expression by desmin suggests that desmin may contribute to cardiogenic commitment and myocardial differentiation by directly influencing the transcription of the nkx2.5 gene in cardiac progenitor cells. Here, we demonstrate that desmin activates transcription of nkx2.5 reporter genes, rescues nkx2.5 haploinsufficiency in cardiac progenitor cells, and is responsible for the proper expression of Nkx2.5 in adult cardiac side population stem cells. These effects are consistent with the temporary presence of desmin in the nuclei of differentiating cardiac progenitor cells and its physical interaction with transcription factor complexes bound to the enhancer and promoter elements of the nkx2.5 gene. These findings introduce desmin as a newly discovered and unexpected player in the regulatory network guiding cardiomyogenesis in cardiac stem cells.

Strategies to Study Desmin in Cardiac Muscle and Culture Systems.

Intermediate filament (IF) cytoskeleton comprises the fine-tuning cellular machinery regulating critical homeostatic mechanisms. In skeletal and cardiac muscle, deficiency or disturbance of the IF network leads to severe pathology, particularly in the latter. The three-dimensional scaffold of the muscle-specific IF protein desmin interconnects key features of the cardiac muscle cells, including the Z-disks, intercalated disks, plasma membrane, nucleus, mitochondria, lysosomes, and potentially sarcoplasmic reticulum. This is crucial for the highly organized striated muscle, in which effective energy production and transmission as well as mechanochemical signaling are tightly coordinated among the organelles and the contractile apparatus. The role of desmin and desmin-associated proteins in the biogenesis, trafficking, and organelle function, as well as the development, differentiation, and survival of the cardiac muscle begins to be enlightened, but the precise mechanisms remain elusive. We propose a set of experimental tools that can be used, in vivo and in vitro, to unravel crucial new pathways by which the IF cytoskeleton facilitates proper organelle function, homeostasis, and cytoprotection and further understand how its disturbance and deficiency lead to disease.

Tumor necrosis factor-α confers cardioprotection through ectopic expression of keratins K8 and K18.

Tumor necrosis factor-α (TNF-α), one of the major stress-induced proinflammatory cytokines, is upregulated in the heart after tissue injury, and its sustained expression can contribute to the development of heart failure. Whether TNF-α also exerts cytoprotective effects in heart failure is not known. Here we provide evidence for a cardioprotective function of TNF-α in a genetic heart failure model, desmin-deficient mice. The cardioprotective effects of TNF-α are a consequence of nuclear factor-κB (NF-κB)-mediated ectopic expression in cardiomyocytes of keratin 8 (K8) and keratin 18 (K18), two epithelial-specific intermediate filament proteins. In cardiomyocytes, K8 and K18 (K8/K18) formed an alternative cytoskeletal network that localized mainly at intercalated discs (IDs) and conferred cardioprotection by maintaining normal ID structure and mitochondrial integrity and function. Ectopic induction of K8/K18 expression in cardiomyocytes also occurred in other genetic and experimental models of heart failure. Loss of the K8/K18 network resulted in a maladaptive cardiac phenotype following transverse aortic constriction. In human failing myocardium, where TNF-α expression is upregulated, K8/K18 were also ectopically expressed and localized primarily at IDs, which did not contain detectable amounts of desmin. Thus, TNF-α- and NF-κB-mediated formation of an alternative, stress-induced intermediate filament cytoskeleton has cardioprotective function in mice and potentially in humans.

Complement system modulation as a target for treatment of arrhythmogenic cardiomyopathy.

Inflammation may contribute to disease progression in arrhythmogenic cardiomyopathy (ACM). However, its role in this process is unresolved. Our goal was to delineate the pathogenic role of the complement system in a new animal model of ACM and in human disease. Using cardiac histology, echocardiography, and electrocardiography, we have demonstrated that the desmin-null mouse (Des-/-) recapitulates most of the pathognomonic features of human ACM. Massive complement activation was observed in the Des-/- myocardium in areas of necrotic cells debris and inflammatory infiltrate. Analysis of C5aR-/-Des-/- double-null animals and a pharmaceutical approach using a C5a inhibitor were used to delineate the pathogenic role of the complement system in the disease progression. Our findings indicate that inhibiting C5aR (CD88) signaling improves cardiac function, histopathology, arrhythmias, and survival after endurance. Containment of the inflammatory reaction at the initiation of cardiac tissue injury (2-3 weeks of age), with consequently reduced myocardial remodeling and the absence of a direct long-lasting detrimental effect of C5a-C5aR signaling on cardiomyocytes, could explain the beneficial action of C5aR ablation in Des-/- cardiomyopathy. We extend the relevance of these findings to human pathophysiology by showing for the first time significant complement activation in the cardiac tissues of patients with ACM, thus suggesting that complement modulation could be a new therapeutic target for ACM.

Desmin related disease: a matter of cell survival failure.

Maintenance of the highly organized striated muscle tissue requires a cell-wide dynamic network that through interactions with all vital cell structures, provides an effective mechanochemical integrator of morphology and function, absolutely necessary for intra-cellular and intercellular coordination of all muscle functions. A good candidate for such a system is the desmin intermediate filament cytoskeletal network. Human desmin mutations and post-translational modifications cause disturbance of this network, thus leading to loss of function of both desmin and its binding partners, as well as potential toxic effects of the formed aggregates. Both loss of normal function and gain of toxic function are linked to mitochondrial defects, cardiomyocyte death, muscle degeneration and development of skeletal myopathy and cardiomyopathy.

Μyospryn: a multifunctional desmin-associated protein.

Desmin, the muscle-specific intermediate filament protein, forms a 3D scaffold that links the contractile apparatus to the costameres of plasma membrane, intercalated disks, the nucleus, and also other membranous organelles. The cellular scaffold formed by desmin and its binding partners might be implicated in signaling and trafficking processes, vital mechanisms for the survival of the mammalian cell. One novel desmin-associated protein is the tripartite motif-like protein myospryn. Myospryn was initially identified as an associated partner to the biogenesis of lysosome-related organelles complex 1 protein dysbindin, implicating its potential involvement in vesicle trafficking and organelle biogenesis and/or positioning. Myospryn is also an A kinase anchoring protein, raising the possibility that together with desmin and other cytoskeletal and signaling proteins, it could participate in the subcellular targeting of protein kinase A activity in striated muscle. As with desmin, different members of this scaffold might play a crucial role in the pathogenesis of muscle disease, since any disturbance in these highly coordinated signaling pathways is expected to compromise efficient maintenance of structure-function integrity of muscle and lead to different cardiac and skeletal myopathies.

Regulation of adverse remodelling by osteopontin in a genetic heart failure model.

AIMS: Desmin, the muscle-specific intermediate filament protein, is a major target in dilated cardiomyopathy and heart failure in humans and mice. The hallmarks of desmin-deficient (des(-/-)) mice pathology include pronounced myocardial degeneration, extended fibrosis, and osteopontin (OPN) overexpression. We sought to identify the molecular and cellular events regulating adverse cardiac remodelling in des(-/-) mice and their potential link to OPN. METHODS AND RESULTS: 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, sustained by innate immunity mechanisms, predominated among all changes occurring in degenerating des(-/-) myocardium. The expression of the most highly up-regulated genes (OPN: 226×, galectin-3: 26×, osteoactivin/Gpnmb/DC-HIL: 160× and metalloprotease-12: 98×) was associated with heart infiltrating macrophages. To evaluate the role of OPN, we generated des(-/-)OPN(-/-) mice and compared their cardiac function and remodelling indices with those of des(-/-). Osteopontin promoted cardiac dysfunction in this model since des(-/-)OPN(-/-) mice showed 53% improvement of left ventricular function, paralleled to an up to 44% reduction in fibrosis. The diminished fibrotic response in the absence of OPN could be partly mediated by a dramatic reduction in myocardial galectin-3 levels, associated with an impaired galectin-3 secretion by OPN-deficient infiltrating macrophages. CONCLUSION: Cardiomyocyte death due to desmin deficiency leads to inflammation and subsequent overexpression of a series of remodelling modulators. Among them, OPN seems to be a major regulator of des(-/-) adverse myocardial remodelling and it functions at least by potentiating galectin-3 up-regulation and secretion.

Osteopontin as a novel prognostic marker in stable ischaemic heart disease: a 3-year follow-up study.

OBJECTIVES: Osteopontin (OPN) is a glycoprotein, which may play a major role in the regulation of biological phenomena. Increased levels of OPN have been linked to the presence and to the severity of atherosclerosis. This study was undertaken to assess the prognostic significance of plasma OPN levels in patients with stable ischaemic heart disease (IHD). METHODS: In 101 patients with stable IHD and angiographically documented significant coronary artery stenosis, plasma OPN levels were measured at baseline (time of coronary arteriography). Patients were prospectively followed for a median time of 3 years (minimum 2.25, maximum 3.9 years). The primary study endpoint was the composite of cardiovascular death, non-fatal myocardial infarction, need for revascularization and hospitalization for cardiovascular reasons. RESULTS: Baseline lnOPN levels were directly related to age (r = 0.27, P < 0.001) and inversely to left ventricular ejection fraction (r = -0.32, P < 0.01). Left ventricular ejection fraction was an independent predictor of plasma OPN levels after adjustment for age and gender (beta = -0.013, P = 0.02). Median OPN value was 55 ng mL(-1). In the univariate Cox-regression analysis, OPN levels > 55 ng mL(-1) (n = 50) were significantly related to adverse cardiac outcome (HR = 2.40, 95% CI: 1.11-5.23, P = 0.027). In multivariate model, OPN levels > 55 ng mL(-1) remained statistically significant independent predictor of adverse outcome after adjustment for age, gender, left ventricular ejection fraction and the number of diseased coronary arteries (HR = 2.88, 95% CI: 1.09-7.58, P = 0.032). CONCLUSION: OPN may provide significant prognostic information independent of other traditional prognostic markers in patients with stable IHD.