Unraveling Desmin's Head Domain Structure and Function.

Understanding the structure and function of intermediate filaments (IFs) is necessary in order to explain why more than 70 related IF genes have evolved in vertebrates while maintaining such dramatically tissue-specific expression. Desmin is a member of the large multigene family of IF proteins and is specifically expressed in myocytes. In an effort to elucidate its muscle-specific behavior, we have used a yeast two-hybrid system in order to identify desmin's head binding partners. We described a mitochondrial and a lysosomal protein, NADH ubiquinone oxidoreductase core subunit S2 (NDUFS2), and saposin D, respectively, as direct desmin binding partners. In silico analysis indicated that both interactions at the atomic level occur in a very similar way, by the formation of a three-helix bundle with hydrophobic interactions in the interdomain space and hydrogen bonds at R16 and S32 of the desmin head domain. The interactions, confirmed also by GST pull-down assays, indicating the necessity of the desmin head domain and, furthermore, point out its role in function of mitochondria and lysosomes, organelles which are disrupted in myopathies due to desmin head domain mutations.

The Han:SPRD Rat: A Preclinical Model of Polycystic Kidney Disease.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) stands as the most prevalent hereditary renal disorder in humans, ultimately culminating in end-stage kidney disease. Animal models carrying mutations associated with polycystic kidney disease have played an important role in the advancement of ADPKD research. The Han:SPRD rat model, carrying an R823W mutation in the Anks6 gene, is characterized by cyst formation and kidney enlargement. The mutated protein, named Samcystin, is localized in cilia of tubular epithelial cells and seems to be involved in cystogenesis. The homozygous Anks6 mutation leads to end-stage renal disease and death, making it a critical factor in kidney development and function. This review explores the utility of the Han:SPRD rat model, highlighting its phenotypic similarity to human ADPKD. Specifically, we discuss its role in preclinical trials and its importance for investigating the pathogenesis of the disease and developing new therapeutic approaches.

PPARδ activation improves cardiac mitochondrial homeostasis in desmin deficient mice but does not alleviate systolic dysfunction.

Peroxisome proliferator-activated receptor (PPAR) δ is a major transcriptional regulator of cardiac energy metabolism with pleiotropic properties, including anti-inflammatory, anti-oxidative and cardioprotective action. In this study, we sought to investigate whether pharmacological activation of PPARδ via intraperitoneal administration of the selective ligand GW0742 could ameliorate heart failure and mitochondrial dysfunction that have been previously reported in a characterized genetic model of heart failure, the desmin null mice (Des-/-). Our studies demonstrate that treatment of Des-/- mice with the PPARδ agonist attenuated cardiac inflammation, fibrosis and cardiac remodeling. In addition, PPARδ activation alleviated oxidative stress in the failing myocardium as evidenced by decreased ROS levels. Importantly, PPARδ activation stimulated mitochondrial biogenesis, prevented mitochondrial and sarcoplasmic reticulum vacuolar degeneration and improved the mitochondrial intracellular distribution. Finally, PPARδ activation alleviated the mitochondrial respiratory dysfunction, prevented energy depletion and alleviated excessive autophagy and mitophagy in Des-/- hearts. Nevertheless, improvement of all these parameters did not suffice to overcome the significant structural deficiencies that desmin deletion incurs in cardiomyocytes and cardiac function did not improve significantly. In conclusion, pharmacological PPARδ activation in Des-/- hearts exerts protective effects during myocardial degeneration and heart failure by preserving the function and quality of the mitochondrial network. These findings implicate PPARδ agonists as a supplemental constituent of heart failure medications.

Return of the Tbx5; lineage-tracing reveals ventricular cardiomyocyte-like precursors in the injured adult mammalian heart.

The single curative measure for heart failure patients is a heart transplantation, which is limited due to a shortage of donors, the need for immunosuppression and economic costs. Therefore, there is an urgent unmet need for identifying cell populations capable of cardiac regeneration that we will be able to trace and monitor. Injury to the adult mammalian cardiac muscle, often leads to a heart attack through the irreversible loss of a large number of cardiomyocytes, due to an idle regenerative capability. Recent reports in zebrafish indicate that Tbx5a is a vital transcription factor for cardiomyocyte regeneration. Preclinical data underscore the cardioprotective role of Tbx5 upon heart failure. Data from our earlier murine developmental studies have identified a prominent unipotent Tbx5-expressing embryonic cardiac precursor cell population able to form cardiomyocytes, in vivo, in vitro and ex vivo. Using a developmental approach to an adult heart injury model and by employing a lineage-tracing mouse model as well as the use of single-cell RNA-seq technology, we identify a Tbx5-expressing ventricular cardiomyocyte-like precursor population, in the injured adult mammalian heart. The transcriptional profile of that precursor cell population is closer to that of neonatal than embryonic cardiomyocyte precursors. Tbx5, a cardinal cardiac development transcription factor, lies in the center of a ventricular adult precursor cell population, which seems to be affected by neurohormonal spatiotemporal cues. The identification of a Tbx5-specific cardiomyocyte precursor-like cell population, which is capable of dedifferentiating and potentially deploying a cardiomyocyte regenerative program, provides a clear target cell population for translationally-relevant heart interventional studies.

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.

Crosstalk between coagulation and complement activation promotes cardiac dysfunction in arrhythmogenic right ventricular cardiomyopathy.

Aims: We previously found that complement components are upregulated in the myocardium of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC), and inhibiting the complement receptor C5aR reduces disease severity in desmin knockout (Des-/- ) mice, a model for ARVC. Here, we examined the mechanism underlying complement activation in ARVC, revealing a potential new therapeutic target. Methods: First, immunostaining, RT-PCR and western blot were used to detect the expression levels of complement and coagulation factors. Second, we knocked out the central complement component C3 in Des-/- mice (ARVC model) by crossing Des-/- mice with C3-/- mice to explore whether complement system activation occurs independently of the conventional pathway. Then, we evaluated whether a targeted intervention to coagulation system is effective to reduce myocardium injury. Finally, the plasma sC5b9 level was assessed to investigate the role in predicting adverse cardiac events in the ARVC cohort. Results: The complement system is activated in the myocardium in ARVC. Autoantibodies against myocardial proteins provided a possible mechanism underlying. Moreover, we found increased levels of myocardial C5 and the serum C5a in Des-/-C3-/- mice compared to wild-type mice, indicating that C5 is activated independently from the conventional pathway, presumably via the coagulation system. Crosstalk between the complement and coagulation systems exacerbated the myocardial injury in ARVC mice, and this injury was reduced by using the thrombin inhibitor lepirudin. In addition, we found significantly elevated plasma levels of sC5b9 and thrombin in patients, and this increase was correlated with all-cause mortality. Conclusions: These results suggest that crosstalk between the coagulation and complement systems plays a pathogenic role in cardiac dysfunction in ARVC. Thus, understanding this crosstalk may have important clinical implications with respect to diagnosing and treating ARVC.

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.

Desmin is essential for the structure and function of the sinoatrial node: implications for increased arrhythmogenesis.

Our objective was to investigate the effect of desmin depletion on the structure and function of the sinoatrial pacemaker complex (SANcl) and its implication in arrhythmogenesis. Analysis of mice and humans (SANcl) indicated that the sinoatrial node exhibits high amounts of desmin, desmoplakin, N-cadherin, and ß-catenin in structures we call "lateral intercalated disks" connecting myocytes side by side. Examination of the SANcl from an arrhythmogenic cardiomyopathy model, desmin-deficient (Des-/-) mouse, by immunofluorescence, ultrastructural, and Western blot analysis showed that the number of these lateral intercalated disks was diminished. Also, electrophysiological recordings of the isolated compact sinoatrial node revealed increased pacemaker systolic potential and higher diastolic depolarization rate compared with wild-type mice. Prolonged interatrial conduction expressed as a longer P wave duration was also observed in Des-/- mice. Upregulation of mRNA levels of both T-type Ca2+ current channels, Cav3.1 and Cav3.2, in the Des-/- myocardium (1.8- and 2.3-fold, respectively) and a 1.9-fold reduction of funny hyperpolarization-activated cyclic nucleotide-gated K+ channel 1 could underlie these functional differences. To investigate arrhythmogenicity, electrocardiographic analysis of Des-deficient mice revealed a major increase in supraventricular and ventricular ectopic beats compared with wild-type mice. Heart rate variability analysis indicated a sympathetic predominance in Des-/- mice, which may further contribute to arrhythmogenicity. In conclusion, our results indicate that desmin elimination leads to structural and functional abnormalities of the SANcl. These alterations may be enhanced by the sympathetic component of the cardiac autonomic nervous system, which is predominant in the desmin-deficient heart, thus leading to increased arrhythmogenesis.NEW & NOTEWORTHY The sinoatrial node exhibits high amounts of desmin and desmoplakin in structures we call "lateral intercalated disks," connecting side-by-side adjacent cardiomyocytes. These structures are diminished in desmin-deficient mouse models. Misregulation of T-type Ca2+ current and hyperpolarization-activated cyclic nucleotide-gated K+ channel 1 was proved along with prolonged interatrial conduction and cardiac autonomic nervous system dysfunction.

Implications of the mitochondrial interactome of mammalian thioredoxin 2 for normal cellular function and disease.

Multiple thioredoxin isoforms exist in all living cells. To explore the possible functions of mammalian mitochondrial thioredoxin 2 (Trx2), an interactome of mouse Trx2 was initially created using (i) a monothiol mouse Trx2 species for capturing protein partners from different organs and (ii) yeast two hybrid screens on human liver and rat brain cDNA libraries. The resulting interactome consisted of 195 proteins (Trx2 included) plus the mitochondrial 16S RNA. 48 of these proteins were classified as mitochondrial (MitoCarta2.0 human inventory). In a second step, the mouse interactome was combined with the current four-membered mitochondrial sub-network of human Trx2 (BioGRID) to give a 53-membered human Trx2 mitochondrial interactome (52 interactor proteins plus the mitochondrial 16S RNA). Although thioredoxins are thiol-employing disulfide oxidoreductases, approximately half of the detected interactions were not due to covalent disulfide bonds. This finding reinstates the extended role of thioredoxins as moderators of protein function by specific non-covalent, protein-protein interactions. Analysis of the mitochondrial interactome suggested that human Trx2 was involved potentially in mitochondrial integrity, formation of iron sulfur clusters, detoxification of aldehydes, mitoribosome assembly and protein synthesis, protein folding, ADP ribosylation, amino acid and lipid metabolism, glycolysis, the TCA cycle and the electron transport chain. The oxidoreductase functions of Trx2 were verified by its detected interactions with mitochondrial peroxiredoxins and methionine sulfoxide reductase. Parkinson's disease, triosephosphate isomerase deficiency, combined oxidative phosphorylation deficiency, and lactate dehydrogenase b deficiency are some of the diseases where the proposed mitochondrial network of Trx2 may be implicated.

Increased expression of Toll-like receptors 2, 3, 4 and 7 mRNA in the kidney and intestine of a septic mouse model.

Toll-like receptors (TLRs) are the key regulators of innate and adaptive immunity and are highly expressed during sepsis. Thus, studying the expression of TLRs in an animal septic model might indicate their possible association with acute kidney injury in sepsis. Seventy-two male C57BL/6J mice were used for this study. Randomly, these animals were divided into 6 groups (N = 12/group): 3 control and 3 septic groups depending on the euthanasia time (24 h, 48 h, 72 h). Septic groups underwent cecal ligation and puncture (CLP) to induce peritonitis, while control groups had a sham operation. Hematological tests were performed in serum for immune biomarkers; immunohistochemistry, morphometry and qRT-PCR analysis were used on both kidney and intestine tissues to evaluate the expression of TLR 2, 3, 4 and 7 in a septic process. At the end of each experimental period, we found that TLRs 2, 3, 4 and 7 were expressed in both tissues but there were differences between those at various time points. Also, we found that mRNA levels were significantly higher in qRT-PCR evaluation in septic groups than control groups in both kidney and intestinal tissues (p < 0.05); showing a steady increase in the septic groups as the time to euthanasia was prolonged (p < 0.05). Overall, our study provides a suggestion that TLRs 2, 3, 4 and 7 are highly expressed in the kidneys of septic mice and especially that these TLRs are sensitive and specific markers for sepsis. Finally, our study supports the diagnostic importance of TLRs in AKI and provides an insight on the contribution of septic mice models in the study of multi organ dysfunction syndrome in general.

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.

Expression of Toll-like receptors (TLRs) in the lungs of an experimental sepsis mouse model.

BACKGROUND: Sepsis is a condition characterized by high mortality rates and often accompanied by multiple-organ dysfunction. During sepsis, respiratory system may be affected and possibly result in acute respiratory distress syndrome (ARDS). Toll-like receptors (TLRs), as a first line defense against invading pathogens, seem to be highly expressed in septic states. Therefore, expression of TLRs in the lungs of a sepsis animal model could indicate the involvement of the respiratory system and appear as a severity index of the clinical course. MATERIALS AND METHODS: A total of 72 C57BL/6J mice, aged 12-14 weeks, were studied. The animals were divided into 3 sepsis (S) groups (24h, 48h and 72h) and 3 control (C) groups (24h, 48h and 72h), each consisting of 12 mice. The S-groups were subjected to cecal ligation and puncture (CLP) while the C-groups had a sham operation performed. Blood samples were drawn from all groups. Total blood count analysis was performed along with the measurement of certain biochemical markers. Additionally, lung tissues were harvested and the expression of TLRs, namely TLR 2, TLR 3, TLR 4 and TLR 7 were evaluated by means of immunofluorescence (IF) and qRT-PCR (quantitative-Polymerase Chain Reaction). Statistical analysis was performed by using one-way ANOVA followed by student t-test. Results were considered statistically significant when p<0.05. RESULTS: WBCs and lymphocytes were decreased in all S-groups compared to the corresponding C-groups (p<0.05), while RBCs showed a gradual decline in S-groups with the lowest levels appearing in the S72 group. Only, monocytes were higher in S-groups, especially between S48-C48 (p<0.05) and S72-C72 (p<0.05). Creatinine, IL-10 and IL-6 levels were significantly increased in the S-groups compared to the corresponding C-groups (S24 vs C24, S48 vs C48 and S72 vs C72, p<0.05). IF showed that expression of TLRs 2, 3, 4 and 7 was increased in all S-groups compared to the time-adjusted C-groups (p<0.05). Similarly, qRT-PCR revealed that expression of all TLRs was higher in all S-groups compared to their respective C-groups in both lungs and intestine (p<0.05). Comparing lung and intestinal tissues from S-groups, TLRs 2 and 4 were found increased in the lung at 24, 48 and 72 hours (p<0.05), whereas TLR 3 was higher in the intestine at all time points examined (p<0.05). Finally, TLR 7 levels were significantly higher in the intestinal tissues at 24 hours (p<0.0001), while lungs predominated at 48 hours (p<0.0001). CONCLUSION: TLRs seem to be highly expressed in the lungs of septic mice, therefore suggesting a potential role in the pathogenesis of ARDS during sepsis. While more studies need to be conducted in order to completely understand the underlying mechanisms, TLRs may represent a promising target for establishing novel therapeutic strategies in the treatment of sepsis.

The neuroprotective agent Rasagiline mesylate attenuates cardiac remodeling after experimental myocardial infarction.

AIM: Rasagiline mesylate (N-propargyl-1 (R)-aminoindan) (RG) is a selective, potent irreversible inhibitor of monoamine oxidase-B with cardioprotective and anti-apoptotic properties. We investigated whether it could be cardioprotective in a rat model undergoing experimental myocardial infarction (MI) by permanent ligation of the left anterior descending coronary artery. METHODS AND RESULTS: RG was administered, intraperitoneally, for 28 days (2 mg/kg) starting 24 h after MI induction. Echocardiography analysis revealed a significant reduction in left ventricular end-systolic and diastolic dimensions and preserved fractional shortening in RG-treated compared with normal saline group at 28 days post-MI (31.6 ± 2.3 vs. 19.6 ± 1.8, P < 0.0001), respectively. Treatment with RG prevented tissue fibrosis as indicated by interstitial collagen estimation by immunofluorescence staining and hydroxyproline content and attenuated the number of apoptotic myocytes in the border zone (65%) as indicated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Caspase 3 relative protein levels were significantly decreased in the non-infarcted myocardium. Markedly decreased malondialdehyde levels in the border zone indicate a reduction in tissue oxidative stress. CONCLUSIONS: Our study demonstrates a positive effect of RG in the post-MI period with a significant attenuation in cardiac remodelling.

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.

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.

Complement system activation in cardiac and skeletal muscle pathology: friend or foe?

A major goal in current cardiology practice is to determine optimal strategies for minimizing myocardial necrosis and optimizing cardiac repair following an acute myocardial infarction. Temporally regulated activation and suppression of innate immunity may be critical for achieving this goal. Extensive experimental data in various animal models have indicated that inhibiting complement activation offers protection to cardiac tissue after ischemia/reperfusion. However, the results of clinical studies using complement inhibitors (mainly at the C5 level) in patients with acute myocardial infarction have largely been disappointing. In cases in which complement activation participates in the initial events of muscle cell destruction, as in autoimmune myocarditis or autoimmune muscle disorders, inhibition of complement activation is expected to prove a successful treatment. In other pathologic conditions in which complement is recruited by degenerating or dying muscle cells, as in ischemia, the ideal approach is probably to modulate rather than abruptly blunt complement activation. Beneficial effects of complement action with regard to waste disposal, recruitment of stem cells, regeneration, angiogenesis, and better utilization of energy sources under hypoxic conditions may also prove important for successful disease treatment. Patient outcome after myocardial infarction almost certainly depend upon the combined activation of several distinct but potentially interrelated signaling pathways, suggesting that a combination of treatments targeted to different pathways should be the therapy of choice, and modulation of complement could be one of them.

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.

Muscle LIM protein interacts with cofilin 2 and regulates F-actin dynamics in cardiac and skeletal muscle.

The muscle LIM protein (MLP) and cofilin 2 (CFL2) are important regulators of striated myocyte function. Mutations in the corresponding genes have been directly associated with severe human cardiac and skeletal myopathies, and aberrant expression patterns have often been observed in affected muscles. Herein, we have investigated whether MLP and CFL2 are involved in common molecular mechanisms, which would promote our understanding of disease pathogenesis. We have shown for the first time, using a range of biochemical and immunohistochemical methods, that MLP binds directly to CFL2 in human cardiac and skeletal muscles. The interaction involves the inter-LIM domain, amino acids 94 to 105, of MLP and the amino-terminal domain, amino acids 1 to 105, of CFL2, which includes part of the actin depolymerization domain. The MLP/CFL2 complex is stronger in moderately acidic (pH 6.8) environments and upon CFL2 phosphorylation, while it is independent of Ca(2+) levels. This interaction has direct implications in actin cytoskeleton dynamics in regulating CFL2-dependent F-actin depolymerization, with maximal depolymerization enhancement at an MLP/CFL2 molecular ratio of 2:1. Deregulation of this interaction by intracellular pH variations, CFL2 phosphorylation, MLP or CFL2 gene mutations, or expression changes, as observed in a range of cardiac and skeletal myopathies, could impair F-actin depolymerization, leading to sarcomere dysfunction and disease.

A missense mutation in desmin tail domain linked to human dilated cardiomyopathy promotes cleavage of the head domain and abolishes its Z-disc localization.

A missense mutation (Ile 451 to Met) at the tail domain of the muscle-specific intermediate filament protein desmin has been suggested to be a genetic cause of dilated cardiomyopathy. The Ile451Met mutation is located inside a conserved motif in the desmin tail domain, believed to have a potential role in the lateral packing of type III intermediate filaments. Nevertheless, the role of the type III intermediate filament tail domain remains elusive. To further study the role of this domain in the function of cardiomyocytes and in the development of cardiomyopathy, we generated transgenic mice expressing the mutant desmin(I451M) in the cardiac tissue. Analysis of hearts from transgenic animals revealed that mutant desmin loses its Z-disc localization but it can still associate with the intercalated discs, which, however, have an altered architecture, resembling other examples of dilated cardiomyopathy. This is the first report demonstrating a critical role of the desmin head and tail domains in the formation of the IF scaffold around Z discs. It is further suggested that in cardiomyocytes, an interplay between desmin tail and head domains is taking place, which potentially protects the amino terminus of desmin from specific proteases. The fact that the association with intercalated discs seems unchanged suggests that this association must take place through the desmin tail, in contrast to the head domain that is most possibly involved in the Z-disc binding.