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.

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.

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.

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.

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.

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.

Cytokeratin 18 as a Novel Biomarker in Patients with Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a heart muscle disease associated with an increased risk for sudden cardiac death (SCD). Cytokeratin 18-based proteins, such as M30 and M65 antigens, are known cell-death biomarkers. M30 antigen is released from cells during apoptosis, and M65 antigen is released during cell death from any cause, such as apoptosis or necrosis. We aimed to study the expression of M30 and M65 antigens in peripheral blood obtained by 46 HCM patients and compare with 27 age- and sex-matched patients without HCM. We also investigated the CK18 expression in myocardium from postmortem HCM hearts. M30 and M65 antigens were significantly increased in the HCM vs. non-HCM group (Μ30: 338 ± 197 U/uL vs. 206 ± 166 U/uL, p = 0.003; M65: 428 ± 224 U/uL vs. 246 ± 214 U/uL, p = 0.001), and HCM patients with a higher expression of these markers (M30: 417 ± 208 vs. 271 ± 162 U/uL, p = 0.011; M65: 518 ± 242 vs. 351 ± 178 U/uL, p = 0.011) had a higher risk for SCD. In HCM, both apoptosis and necrosis are increased, but particularly necrosis (M30/M65 ratio: 0.75 ± 0.09 vs. 0.85 ± 0.02, p < 0.001). CK18 is expressed in the HCM myocardium (1.767 ± 0.412 vs. 0.537 ± 0.383, % of area, p = 0.0058). Therefore, M30 and M65 antigens may be novel biomarkers in HCM.

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.