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Viral myocarditis (VM) is an inflammatory pathology of the myocardium triggered by a viral infection that may cause sudden death or heart failure, especially in the younger population. Current treatments only stabilise and improve cardiac function without resolving the underlying inflammatory cause. The factors that induce VM to progress to heart failure are still uncertain, but neutrophils have been increasingly associated with the negative evolution of cardiac pathologies. The present study investigates the contribution of neutrophils to VM disease progression in different ways.In a Coxsackievirus B3- (CVB3) induced mouse model of VM, neutrophils and neutrophil extracellular traps (NETs) were prominent in the acute phase of VM as revealed by ELISA analysis and immunostaining. Anti-Ly6G-mediated neutrophil blockade starting at model induction decreased cardiac necrosis and leukocyte infiltration, preventing monocyte and Ly6CHigh pro-inflammatory macrophage recruitment. Furthermore, genetic peptidylarginine deiminase 4 (PAD4)-dependent NET blockade significantly reduced cardiac damage and leukocyte recruitment, significantly decreasing cardiac monocyte and macrophage presence. Depleting neutrophils with anti-Ly6G antibodies at 7 days post-infection, after the acute phase, did not decrease cardiac inflammation.Collectively, these results indicate that the repression of neutrophils and the related NET response in the acute phase of VM improves the pathological phenotype by reducing cardiac inflammation.Viral myocarditis (VM) is a form of acute heart failure prompted by viral infection that still lacks a specific therapy addressing the cardiac inflammation causing the disease. Increasing evidence suggests that neutrophils actively contribute to the severity of inflammatory and cardiovascular pathologies. Our study demonstrates that inhibition of neutrophil functions in the early phases of VM decreases cardiac damage and inflammation and, therefore, may be considered a very early therapeutic strategy in preventing disease progression.

Pathological innate and adaptive immune response upon viral infection may lead to cardiac injury and dysfunction. Stabilin-1 is a scavenger receptor that regulates several aspects of the innate immunity. Whether stabilin-1 affects the inflammatory response during viral myocarditis (VM) is entirely unknown. Here, we assess the role of stabilin-1 in the pathogenesis of VM and its suitability as a therapeutic target. Genetic loss of stabilin-1 increased mortality and cardiac necrosis in a mouse model of human Coxsackievirus B3 (CVB3)-induced myocarditis. Absence of stabilin-1 significantly reduced monocyte recruitment and strongly reduced the number of alternatively activated anti-inflammatory macrophages in the heart, enhancing a pro-inflammatory cardiac niche with a detrimental T lymphocyte response during VM. Yeast two-hybrid screening, confirmed by affinity chromatography, identified fibronectin as a stabilin-1 interacting partner. Absence of stabilin-1 specifically decreased monocyte adhesion on extracellular fibronectin in vitro. Loss of Type III repeats Extra Domain A (EDA) of fibronectin during VM also increased the mortality and cardiac necrosis as in stabilin-1 knockout mice, with reduced monocytic cardiac recruitment and increased T lymphocyte response. Collectively, stabilin-1 has an immune-suppressive role of limiting myocardial damage during VM, regulating anti-inflammatory monocyte-recruitment to the site of inflammation. ispartof: Journal Of Molecular And Cellular Cardiology vol:165 pages:31-39 ispartof: location:England status: published

Background Aging can be viewed as a status of chronic inflammation, in which neutrophils have a lower threshold for activation. The enzyme peptidylarginine deiminase 4 (PAD4), which catalyzes the conversion of arginine to citrulline, will be activated in a certain population of neutrophils. When this conversion takes place on the histones, neutrophils can form neutrophil extracellular traps (NETs), which are both prothrombotic and proinflammatory. Mice lacking this enzyme systemically were previously reported to be protected from age-related fibrosis. Purpose We aimed to study the long-term effect of neutrophils on cardiac health during the process of natural aging. We hypothesized that neutrophil PAD4, and in consequence NETs, are involved in cardiac fibrosis development, which in turn will result in impaired cardiac function. Methods We generated a mouse model of impaired NET release capability via deletion of PAD4, a NET-essential gene, under the neutrophil-specific promoter (PAD4fl/flMRP8Cre+). In order to study heart failure (HF) development, these specific deletion mice and their littermate controls were aged for a period of two years (coinciding with approximately 70 years of age in the human population; the age at which HF is the number one cause of hospitalization), after which cardiac function and remodeling were evaluated by echocardiography and histology, respectively. A separate set of young mice (12 weeks) were evaluated in parallel. Results We performed a comprehensive echocardiography analysis including both structural and functional parameters. As for systolic function, we could see that in old wild type (WT) mice, ejection fraction (EF) significantly decreased as compared to EF in young and healthy (YH) mice (YH - 67±6%, WT - 53±10%; p Conclusion Our data confirms neutrophil PAD4 involvement in heart failure progression by promoting cardiac fibrosis, resulting in cardiac dysfunction. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Fonds Wetenschappelijk Onderzoek (FWO) - Vlaanderen

Muscular dystrophies are debilitating neuromuscular disorders for which no cure exists. As this disorder affects both cardiac and skeletal muscle, patients would benefit from a cellular therapy that can simultaneously regenerate both tissues. The current protocol to derive bipotent mesodermal progenitors which can differentiate into cardiac and skeletal muscle relies on the spontaneous formation of embryoid bodies, thereby hampering further clinical translation. Additionally, as skeletal muscle is the largest organ in the human body, a high myogenic potential is necessary for successful regeneration. Here, we have optimized a protocol to generate chemically defined induced pluripotent stem cell-derived mesodermal progenitors (cdMiPs). We demonstrate that these cells contribute to myotube formation and differentiate into cardiomyocytes, both in vitro and in vivo. Furthermore, the addition of valproic acid, a clinically approved small molecule, increases the potential of the cdMiPs to contribute to myotube formation without compromising their ability to differentiate towards cardiomyocytes. This effect is mediated through the activation of the Notch signaling pathway. Taken together, these results constitute a novel approach to generate mesodermal progenitors with enhanced myogenic potential using clinically approved reagents, which opens the door to new therapeutic solutions in the treatment of muscular dystrophy.

The metabolic syndrome (MetS) is an escalating problem worldwide, causing left ventricular stiffening, an early characteristic of diastolic dysfunction for which no treatment exists. As diastolic dysfunction and stiffening in MetS patients are associated with increased circulating dipeptidyl peptidase-4 (DPP-4) levels, we investigated whether the clinically approved DPP-4 inhibitor linagliptin reduces left ventricular stiffness in MetS-induced cardiac disease. Sixteen-week-old obese ZSF1 rats, displaying the MetS and left ventricular stiffness, received linagliptin-supplemented or placebo diet for four weeks. Linagliptin significantly reduced obesity, hyperlipidaemia, and hyperglycaemia and improved left ventricular relaxation. This improved relaxation was related to decreased cardiac fibrosis and cardiomyocyte passive stiffness (Fpassive ). The reduced Fpassive was the result of titin isoform switching from the stiff N2B to the more flexible N2BA and increased phosphorylation of total titin and specifically its N2Bus region (S4080 and S3391). Importantly, DPP-4 directly cleaved titin in vitro, resulting in an increased Fpassive , which was prevented by simultaneous administration of linagliptin. In conclusion, linagliptin improves left ventricular stiffness in obese ZSF1 rats by preventing direct DPP4-mediated titin cleavage, as well as by modulating both titin isoform levels and phosphorylation. Reducing left ventricular stiffness by administering linagliptin might prevent MetS-induced early diastolic dysfunction in human. ispartof: Journal Of Cellular And Molecular Medicine vol:25 issue:2 ispartof: location:England status: Published online

Heart failure with preserved ejection fraction (HFpEF) is currently untreated. Therapeutics development demands effective diagnosis of diastolic dysfunction in animal models mimicking human pathology, which requires appropriate anaesthetics. Here, we investigated which anaesthetic, ketamine/xylazine or isoflurane, could be used to reveal diastolic dysfunction in HFpEF-diseased obese ZSF1 rats by echocardiography. First, diastolic dysfunction was confirmed by pressure-volume loops in obese compared to lean control ZSF1 rats. In echocardiography, ketamine/xylazine, unlike isoflurane, was able to demonstrate impaired relaxation in obese ZSF1 rats, as reflected by impaired early (E) and late (A) filling peak velocities, decreased E/A ratio, and a prolonged deceleration and isovolumic relaxation time. Interestingly, ketamine/xylazine induced a wider separation of both tissue and pulsed wave Doppler-derived echocardiographic waves required for diastolic dysfunction diagnosis, potentially by reducing the heart rate (HR), while isoflurane resulted in merged waves. To assess whether HR-lowering alone explained the differences between the anaesthetics, echocardiography measurements under isoflurane with and without the HR-lowering drug ivabradine were compared. However, diastolic dysfunction could not be diagnosed in ivabradine-treated obese ZSF1 rats. In summary, ketamine/xylazine compared to isoflurane is the anaesthetic of choice to detect diastolic dysfunction by echocardiography in rodent HFpEF, which was only partly mediated by HR-lowering. ispartof: Sci Rep vol:10 issue:1 ispartof: location:England status: Published online

Contractile myofiber units are mainly composed of thick myosin and thin actin (F-actin) filaments. F-Actin interacts with Microtubule Associated Monooxygenase, Calponin And LIM Domain Containing 2 (MICAL2). Indeed, MICAL2 modifies actin subunits and promotes actin filament turnover by severing them and preventing repolymerization. In this study, we found that MICAL2 increases during myogenic differentiation of adult and pluripotent stem cells (PSCs) towards skeletal, smooth and cardiac muscle cells and localizes in the nucleus of acute and chronic regenerating muscle fibers. In vivo delivery of Cas9-Mical2 guide RNA complexes results in muscle actin defects and demonstrates that MICAL2 is essential for skeletal muscle homeostasis and functionality. Conversely, MICAL2 upregulation shows a positive impact on skeletal and cardiac muscle commitments. Taken together these data demonstrate that modulations of MICAL2 have an impact on muscle filament dynamics and its fine-tuned balance is essential for the regeneration of muscle tissues. ispartof: CELL DEATH & DISEASE vol:11 issue:8 ispartof: location:England status: published

The aggrecanase ADAMTS5 (A Disintegrin and Metalloproteinase with ThromboSpondin type 1 motifs, member 5) and the cleavage of its substrate versican have been implicated in the development of heart valves. Furthermore, ADAMTS5 deficiency was shown to protect against diet-induced obesity, a known risk factor for cardiovascular disease. Therefore, in this study, we investigated the potential role of ADAMTS5 in cardiac function using ADAMTS5-deficient (Adamts5-/- ) mice and their wild-type (Adamts5+/+ ) counterparts exposed to a standard-fat or a high-fat diet (HFD). Eight-weeks-old Adamts5-/- and Adamts5+/+ mice were exposed to each diet for 15 weeks. Cardiac function and electrophysiology were analyzed by transthoracic echocardiogram and electrocardiogram at the end of the study. Cleavage of versican, as detected by the appearance of the DPEEAE neo-epitope on western blotting with protein extracts, was defective in the heart of HFD-treated Adamts5-/- as compared with Adamts5+/+ mice. ADAMTS5 deficiency led to statistically significant increases in diastolic posterior wall thickness (0.94 ± 0.023 vs. 0.82 ± 0.036 mm; P = 0.0056) and left ventricle volume (47 ± 4.5 vs. 31 ± 2.5 μL; P = 0.0043) in comparison to Adamts5+/+ mice, but only in animals on a HFD. Cardiac function parameters such as ejection fraction, fractional shortening, and stroke volume were unaffected by ADAMTS5 deficiency or diet. Electrocardiogram analysis revealed no ADAMTS5-specific changes in either diet group. Thus, in the absence of ADAMTS5, cleavage of versican in the cardiac extracellular matrix is impaired, but cardiac function, even upon exposure to a HFD, is not markedly affected. ispartof: CELL BIOLOGY INTERNATIONAL vol:43 issue:6 pages:593-604 ispartof: location:England status: published

MicroRNA-103/107 regulate systemic glucose metabolism and insulin sensitivity. For this reason, inhibitory strategies for these microRNAs are currently being tested in clinical trials. Given the high metabolic demands of the heart and the abundant cardiac expression of miR-103/107, we questioned whether antagomiR-mediated inhibition of miR-103/107 in C57BL/6J mice impacts on cardiac function. Notably, fractional shortening decreased after 6 weeks of antagomiR-103 and -107 treatment. This was paralleled by a prolonged systolic radial and circumferential time to peak and by a decreased global strain rate. Histology and electron microscopy showed reduced cardiomyocyte area and decreased mitochondrial volume and mitochondrial cristae density following antagomiR-103 and -107. In line, antagomiR-103 and -107 treatment decreased mitochondrial OXPHOS complexes’ protein levels compared to scrambled, as assessed by mass spectrometry-based label-free quantitative proteomics. MiR-103/107 inhibition in primary cardiomyocytes did not affect glycolysis rates, but it decreased mitochondrial reserve capacity, reduced mitochondrial membrane potential, and altered mitochondrial network morphology, as assessed by live-cell imaging. Our data indicate that antagomiR-103 and -107 decrease cardiac function, cardiomyocyte size, and mitochondrial oxidative capacity in the absence of pathological stimuli. These data raise concern about the possible cardiac implications of the systemic use of antagomiR-103 and -107 in the clinical setting, and careful cardiac phenotyping within ongoing trials is highly recommended. Keywords: Cardiomyocyte, cardiac, microRNA, miR-103, miR-107, antagomiR, mitochondria, metabolism, mass spectrometry, electron microscopy

Genetic evidence explicitly points out a role for LZTR1, an adaptor for cullin 3 (CUL3) ubiquitin ligase complex, in human disease. Mutations in LZTR1 concurrent with loss of heterozygosity have been associated with glioblastoma and schwannomatosis. LZTR1 emerges as a predisposition gene for paediatric neoplasms. LZTR1 mutations are significantly higher than the background in liver cancers and testicular germ cell tumours. LZTR1 has been also recently added to the list of genes causing Noonan Syndrome, a RASopathy disorder. However, how LZTR1 contributes to human disease and whether it regulates the RAS/MAPK pathway are not known. We found that whereas Lztr1 knockout in mice is embryonically lethal, Lztr1 haploinsufficiency partially recapitulates Noonan syndrome phenotypes, including growth delay, craniofacial dysmorphism, and congenital heart defects. On the other hand, LZTR1 loss in primary Schwann cells drives their dedifferentiation into proliferating cells. By trapping LZTR1 complexes from intact mammalian cells, we identified RAS as a substrate for the LZTR1/CUL3 ubiquitin ligase complex. Immunoprecipitation experiments confirmed that endogenous LZTR1 is associated with endogenous RAS. Ubiquitome and proximity ligation analyses showed that Lztr1 loss abrogates ubiquitination of endogenous RAS specifically at lysine 170. Molecular simulations together with the analysis of Hras-K170R knock-in cells demonstrated that LZTR1-mediated ubiquitination at lysine 170 inhibits RAS signaling by attenuating its association with the membrane. Disease-associated LZTR1 mutations abolish RAS ubiquitination due to disruption of either the LZTR1/CUL3 complex formation, or the interaction with RAS proteins. In line with the work of Bigenzahn et al., Giulio Superti-Furga lab, CeMM (see separate abstract), our study implies LZTR1-mediated RAS ubiquitination as a key step in the control of the RAS GTPases. The discovered molecular mechanism provides a fundamental explanation for the role of LZTR1 in human disease. Citation Format: Mikhail Steklov, Silvia Pandolfi, Maria Francesca Baietti, Paolo Carai, Mingzhen Zhang, Hyunbum Jang, Yanyan Cai, Eric Legius, Jan Tavernier, Kris Gevaert, Francis Impens, Stephane Heymans, Ludwine Messiaen, Ruth Nussimov, Sven Eyckerman, Anna Sablina. Mutations in the ubiquitin ligase adaptor LZTR1 drive human disease by dysregulating RAS ubiquitination and signaling [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A14.

Secreted protein acidic and rich in cysteine (SPARC) is a non-structural extracellular matrix protein that regulates interactions between the matrix and neighboring cells. In the cardiovascular system, it is expressed by cardiac fibroblasts, endothelial cells, and in lower levels by ventricular cardiomyocytes. SPARC expression levels are increased upon myocardial injury and also during hypertrophy and fibrosis. We have previously shown that SPARC improves cardiac function after myocardial infarction by regulating post-synthetic procollagen processing, however whether SPARC directly affects cardiomyocyte contraction is still unknown. In this study we demonstrate a novel inotropic function for extracellular SPARC in the healthy heart as well as in the diseased state after myocarditis-induced cardiac dysfunction. We demonstrate SPARC presence on the cardiomyocyte membrane where it is co-localized with the integrin-beta1 and the integrin-linked kinase. Moreover, extracellular SPARC directly improves cardiomyocyte cell shortening ex vivo and cardiac function in vivo, both in healthy myocardium and during coxsackie virus-induced cardiac dysfunction. In conclusion, we demonstrate a novel inotropic function for SPARC in the heart, with a potential therapeutic application when myocyte contractile function is diminished such as that caused by a myocarditis-related cardiac injury.

Myocardial damage as a consequence of cardiotropic viruses leads to a broad variety of clinical presentations and is still a complicated condition to diagnose and treat. Whereas the extracellular matrix protein Secreted Protein Acidic and Rich in Cysteine or SPARC has been implicated in hypertensive and ischemic heart disease by modulating collagen production and cross-linking, its role in cardiac inflammation and endothelial function is yet unknown. Absence of SPARC in mice resulted in increased cardiac inflammation and mortality, and reduced cardiac systolic function upon coxsackievirus-B3 induced myocarditis. Intra-vital microscopic imaging of the microvasculature of the cremaster muscle combined with electron microscopic imaging of the microvasculature of the cardiac muscle uncovered the significance of SPARC in maintaining endothelial glycocalyx integrity and subsequent barrier properties to stop inflammation. Moreover, systemic administration of recombinant SPARC restored the endothelial glycocalyx and consequently reversed the increase in inflammation and mortality observed in SPARC KO mice in response to viral exposure. Reducing the glycocalyx in vivo by systemic administration of hyaluronidase, an enzyme that degrades the endothelial glycocalyx, mimicked the barrier defects found in SPARC KO mice, which could be restored by subsequent administration of recombinant SPARC. In conclusion, the secreted glycoprotein SPARC protects against adverse cardiac inflammation and mortality by improving the glycocalyx function and resulting endothelial barrier function during viral myocarditis. ispartof: MATRIX BIOLOGY vol:74 pages:21-34 ispartof: location:Netherlands status: published

Diabetic retinopathy (DR) is one of the major complications of diabetes, which eventually leads to blindness. Up to date, no animal model has yet shown all the co-morbidities often observed in DR patients. Here, we investigated whether obese 42 weeks old ZSF1 rat, which spontaneously develops diabetes, hypertension and obesity, would be a suitable model to study DR. Although arteriolar tortuosity increased in retinas from obese as compared to lean (hypertensive only) ZSF1 rats, vascular density pericyte coverage, microglia number, vascular morphology and retinal thickness were not affected by diabetes. These results show that, despite high glucose levels, obese ZSF1 rats did not develop DR. Such observations prompted us to investigate whether the expression of genes, possibly able to contain DR development, was affected. Accordingly, mRNA sequencing analysis showed that genes (i.e. Npy and crystallins), known to have a protective role, were upregulated in retinas from obese ZSF1 rats. Lack of retina damage, despite obesity, hypertension and diabetes, makes the 42 weeks of age ZSF1 rats a suitable animal model to identify genes with a protective function in DR. Further characterisation of the identified genes and downstream pathways could provide more therapeutic targets for the treat DR. ispartof: Scientific Reports vol:8 issue:1 ispartof: location:England status: published

Acute cellular rejection (ACR) is the adverse response of the recipient's immune system against the allogeneic graft. Using human surveillance endomyocardial biopsies (EMBs) manifesting ACR and murine allogeneic grafts, we profiled implicated microRNAs (miRs) and mRNAs. MiR profiling showed that miR‐21, ‐142‐3p, ‐142‐5p, ‐146a, ‐146b, ‐155, ‐222, ‐223, and ‐494 increased during ACR in humans and mice, whereas miR‐149‐5p decreased. mRNA profiling revealed 70 common differentially regulated transcripts, all involved in immune signaling and immune‐related diseases. Interestingly, 33 of 70 transcripts function downstream of IL‐6 and its transcription factor spleen focus forming virus proviral integration oncogene (SPI1), an established target of miR‐155, the most upregulated miR in human EMBs manifesting rejection. In a mouse model of cardiac transplantation, miR‐155 absence and pharmacological inhibition attenuated ACR, demonstrating the causal involvement and therapeutic potential of miRs. Finally, we corroborated our miR signature in acute cellular renal allograft rejection, suggesting a nonorgan specific signature of acute rejection. We concluded that miR and mRNA profiling in human and murine ACR revealed the shared significant dysregulation of immune genes. Inflammatory miRs, for example miR‐155, and transcripts, in particular those related to the IL‐6 pathway, are promising therapeutic targets to prevent acute allograft rejection., The authors identify common mRNA pathways and microRNAs differentially expressed during acute cardiac allograft rejection in mice and humans, and subsequently show that targeting the proinfl ammatory microRNA–155 attenuates inflammation and prolongs graft survival. See also Starling et al on page 121.

Aims Viral myocarditis (VM) is severe cardiac inflammation that can result in sudden death or congestive heart failure in previously healthy adults, with no effective therapy. Liver X receptor (LXR) agonists have both anti-inflammatory and lipid-lowering properties. This study investigates whether LXR agonist T0901317 may modulate viral replication and cardiac inflammation during VM. Methods and results (i) Adult mice were administered T0901317 or vehicle with the onset of inflammation during CVB3 virus myocarditis or (ii) treated 2 days prior to CVB3 infection. Against what we expected, T0901317 treatment did not alter leucocyte infiltration after CVB3 infection; yet pre-administration with T0901317 resulted in increased mortality upon CVB3 infection, higher cardiac viral presence, and increased cardiomyocyte damage when compared with the vehicle. Furthermore, we show a correlation of fatty acid synthase (FAS) and sterol regulatory element-binding protein 1c (SREBP-1c) with CVB3 viral load in the heart and that T0901317 is able to enhance the cardiac expression of FAS and SREBP-1c. Finally, we show in vitro that T0901317 is able to exaggerate CVB3-mediated damage of Vero cells, whereas inhibitors of FAS and the SREBP-1c reduce the viral presence of CVB3 in neonatal cardiomyocytes. Conclusion LXR agonism does not modulate cardiac inflammation, but exacerbates virus-mediated myocardial damage during VM by stimulating lipid biosynthesis and enhancing CVB3 replication.

Regulation of RAS by ubiquitination The protein LZTR1 is mutated in human cancers and developmental diseases. Work from two groups now converges to implicate the protein in regulating signaling by the small guanosine triphosphatase RAS. Steklov et al. showed that mice haploinsufficient for LZTR1 recapitulated aspects of the human disease Noonan syndrome. Their biochemical studies showed that LZTR1 associated with RAS. LZTR1 appears to function as an adaptor that promotes ubiquitination of RAS, thus inhibiting its signaling functions. Bigenzahn et al. found LZTR1 in a screen for proteins whose absence led to resistance to the tyrosine kinase inhibitors used to treat cancers caused by the BCR-ABL oncogene product. Their biochemical studies and genetic studies in fruitflies also showed that loss of LZTR1 led to increased activity of RAS and signaling through the mitogen-activated protein kinase pathway. Science , this issue p. 1177 , p. 1171

Rationale: To maintain cardiac mechanical and structural integrity after an ischemic insult, profound alterations occur within the extracellular matrix. Osteoglycin is a small leucine-rich proteoglycan previously described as a marker of cardiac hypertrophy. Objective: To establish whether osteoglycin may play a role in cardiac integrity and function after myocardial infarction (MI). Methods and Results: Osteoglycin expression is associated with collagen deposition and scar formation in mouse and human MI. Absence of osteoglycin in mice resulted in significantly increased rupture-related mortality with tissue disruption, intramyocardial bleeding, and increased cardiac dysfunction, despite equal infarct sizes. Surviving osteoglycin null mice had greater infarct expansion in comparison with wild-type mice because of impaired collagen fibrillogenesis and maturation in the infarcts as revealed by electron microscopy and collagen polarization. Absence of osteoglycin did not affect cardiomyocyte hypertrophy in the remodeling remote myocardium. In cultured fibroblasts, osteoglycin knockdown or supplementation did not alter transforming growth factor-β signaling. Adenoviral overexpression of osteoglycin in wild-type mice significantly improved collagen quality, thereby blunting cardiac dilatation and dysfunction after MI. In osteoglycin null mice, adenoviral overexpression of osteoglycin was unable to prevent rupture-related mortality because of insufficiently restoring osteoglycin protein levels in the heart. Finally, circulating osteoglycin levels in patients with heart failure were significantly increased in the patients with a previous history of MI compared with those with nonischemic heart failure and correlated with survival, left ventricular volumes, and other markers of fibrosis. Conclusions: Increased osteoglycin expression in the infarct scar promotes proper collagen maturation and protects against cardiac disruption and adverse remodeling after MI. In human heart failure, osteoglycin is a promising biomarker for ischemic heart failure.

Optimal healing after myocardial infarction requires not only the induction of inflammation, but also its timely resolution. In patients, 30 days post myocardial infarction, circulating monocytes have increased expression of Semaphorin3A (Sema3A) as compared to directly after admission. This increased expression coincides with increased expression of Cx3CR1—a marker of non-classical monocytes that are important for immune resolution hence proper wound healing. In mice, the expression of Sema3A also increases in response to myocardial ischemia being expressed by infiltrating leukocytes. Comparing Sema3A heterozygote (HZ) and wild type (WT) mice post myocardial infarction, revealed increased presence of leukocytes in the cardiac tissues of HZ mice as compared to WT, with no differences in capillary density, collagen deposition, cardiomyocyte surface area, chemokine—or adhesion molecules expression. Whilst infarct sizes were similar 14 days after myocardial infarction in both genotypes, Sema3A HZ mice had thinner infarcts and reduced cardiac function as compared to their WT littermates. In vitro experiments were conducted to study the role of Sema3A in inflammation and resolution of inflammation as a potential explanation for the differences in leukocyte recruitment and cardiac function observed in our in vivo experiments. Here, recombinant Sema3A protein was able to affect the pro-inflammatory state of cultured bone marrow derived macrophages. First, the pro-inflammatory state was altered by the induced apoptosis of classical macrophages in the presence of Sema3A. Second, Sema3A promoted the polarization of classical macrophages to resolution-phase macrophages and enhanced their efferocytotic ability, findings that were reflected in the infarcted cardiac tissue of the Sema3A HZ mice. Finally, we demonstrated that besides promoting resolution of inflammation, Sema3A was also able to retard the migration of monocytes to the myocardium. Collectively our data demonstrate that Sema3A reduces cardiac inflammation and improves cardiac function after myocardial infarction by promoting the resolution of inflammation. Electronic supplementary material The online version of this article (doi:10.1007/s00395-017-0630-5) contains supplementary material, which is available to authorized users.

Background: Cardiovascular diseases remain the predominant cause of death worldwide, with the prevalence of heart failure continuing to increase. Despite increased knowledge of the metabolic alterations that occur in heart failure, novel therapies to treat the observed metabolic disturbances are still lacking. Methods: Mice were subjected to pressure overload by means of angiotensin-II infusion or transversal aortic constriction. MicroRNA-146a was either genetically or pharmacologically knocked out or genetically overexpressed in cardiomyocytes. Furthermore, overexpression of dihydrolipoyl succinyltransferase (DLST) in the murine heart was performed by means of an adeno-associated virus. Results: MicroRNA-146a was upregulated in whole heart tissue in multiple murine pressure overload models. Also, microRNA-146a levels were moderately increased in left ventricular biopsies of patients with aortic stenosis. Overexpression of microRNA-146a in cardiomyocytes provoked cardiac hypertrophy and left ventricular dysfunction in vivo, whereas genetic knockdown or pharmacological blockade of microRNA-146a blunted the hypertrophic response and attenuated cardiac dysfunction in vivo. Mechanistically, microRNA-146a reduced its target DLST—the E2 subcomponent of the α-ketoglutarate dehydrogenase complex, a rate-controlling tricarboxylic acid cycle enzyme. DLST protein levels significantly decreased on pressure overload in wild-type mice, paralleling a decreased oxidative metabolism, whereas DLST protein levels and hence oxidative metabolism were partially maintained in microRNA-146a knockout mice. Moreover, overexpression of DLST in wild-type mice protected against cardiac hypertrophy and dysfunction in vivo. Conclusions: Altogether we show that the microRNA-146a and its target DLST are important metabolic players in left ventricular dysfunction.

Background— Cardiac hypertrophy and subsequent heart failure triggered by chronic hypertension represent major challenges for cardiovascular research. Beyond neurohormonal and myocyte signaling pathways, growing evidence suggests inflammatory signaling pathways as therapeutically targetable contributors to this process. We recently reported that microRNA-155 is a key mediator of cardiac inflammation and injury in infectious myocarditis. Here, we investigated the impact of microRNA-155 manipulation in hypertensive heart disease. Methods and Results— Genetic loss or pharmacological inhibition of the leukocyte-expressed microRNA-155 in mice markedly reduced cardiac inflammation, hypertrophy, and dysfunction on pressure overload. These alterations were macrophage dependent because in vivo cardiomyocyte-specific microRNA-155 manipulation did not affect cardiac hypertrophy or dysfunction, whereas bone marrow transplantation from wild-type mice into microRNA-155 knockout animals rescued the hypertrophic response of the cardiomyocytes and vice versa. In vitro, media from microRNA-155 knockout macrophages blocked the hypertrophic growth of stimulated cardiomyocytes, confirming that macrophages influence myocyte growth in a microRNA-155 -dependent paracrine manner. These effects were at least partly mediated by the direct microRNA-155 target suppressor of cytokine signaling 1 (Socs1) because Socs1 knockdown in microRNA-155 knockout macrophages largely restored their hypertrophy-stimulating potency. Conclusions— Our findings reveal that microRNA-155 expression in macrophages promotes cardiac inflammation, hypertrophy, and failure in response to pressure overload. These data support the causative significance of inflammatory signaling in hypertrophic heart disease and demonstrate the feasibility of therapeutic microRNA targeting of inflammation in heart failure.

Rationale: Viral myocarditis results from an adverse immune response to cardiotropic viruses, which causes irreversible myocyte destruction and heart failure in previously healthy people. The involvement of microRNAs and their usefulness as therapeutic targets in this process are unknown. Objective: To identify microRNAs involved in viral myocarditis pathogenesis and susceptibility. Methods and Results: Cardiac microRNAs were profiled in both human myocarditis and in Coxsackievirus B3-injected mice, comparing myocarditis-susceptible with nonsusceptible mouse strains longitudinally. MicroRNA responses diverged depending on the susceptibility to myocarditis after viral infection in mice. MicroRNA-155, -146b, and -21 were consistently and strongly upregulated during acute myocarditis in both humans and susceptible mice. We found that microRNA-155 expression during myocarditis was localized primarily in infiltrating macrophages and T lymphocytes. Inhibition of microRNA-155 by a systemically delivered LNA-anti-miR attenuated cardiac infiltration by monocyte-macrophages, decreased T lymphocyte activation, and reduced myocardial damage during acute myocarditis in mice. These changes were accompanied by the derepression of the direct microRNA-155 target PU.1 in cardiac inflammatory cells. Beyond the acute phase, microRNA-155 inhibition reduced mortality and improved cardiac function during 7 weeks of follow-up. Conclusions: Our data show that cardiac microRNA dysregulation is a characteristic of both human and mouse viral myocarditis. The inflammatory microRNA-155 is upregulated during acute myocarditis, contributes to the adverse inflammatory response to viral infection of the heart, and is a potential therapeutic target for viral myocarditis.

Hypertension and/or aging impose stress on the heart with consecutive cardiac hypertrophy, inflammation and fibrosis, processes that lead to heart failure. Our group has previously shown that non-structural matrix proteins such as TSP-2 and SPARC are critical during hypertension, fibrosis, and age-related cardiac dysfunction. Osteoglycin (OGN) is a class III member of the small leucine rich proteoglycans, and was recently identified as a marker of increased left ventricular mass in a study using genomics in recombinant inbred hypertensive rat strains. Its precise biological role in cardiac remodeling, however, remains unclear. We hypothesize that OGN will influence cardiac hypertrophy and fibrosis and hence is critical in the cardiac response during hypertension and aging. We show in young adult mice (3 months old) that hypertension-induced pressure overload of the heart causes two phases of cardiac fibrosis, following 28 days of angiotensin-II (AngII) infusion. The first phase develops between days 3 to 7 after the AngII-treatment and consists of diffuse interstitially deposited loose fibers. During the second wave of fibrosis, which starts at 14 days, these loose fibers transition into more localized scars of denser collagens. Interestingly, the expression of OGN mRNA also shows a biphasic response, significantly decreasing prior to each phase of fibrosis, at day 1 (96% decrease, p=0.002) and day 14 (84% decrease, p=0.004) respectively. Moreover, hypertension causes exaggerated cardiac fibrosis (7.54±1.58 vs. 3.65±0.62 %, p=0.04) and increased heart weight to body weight ratios (6.39±0.31 vs. 5.42±0.17 mg/g, p=0.01) in young adult OGN null mice when compared to WT littermates, but no differences in the amount of infiltrating leukocytes (31.48±9.82 vs. 36.32±18.75 cells/mm2), after 28 days of AngII infusion. In addition, aging results in decreased cardiac function, as measured by ejection fraction, in 18 months versus 3 months old OGN null mice (42.65±2.74 vs. 58.05±1.37 %, p=0.0001), which is absent in WT mice (51.01±3.11 vs. 53.01±1.67 %, p=0.62). Furthermore, a follow-up study in WT and OGN null mice showed a decrease in stroke volume over time in the OGN null mice (39.30±2.90 μl at 6 months, 35.54±1.95 μl at 12 months and 31.13±1.29 μl at 18 months, p=0.04 between 6 and 18 months) but not in the WT mice (41.61±2.02 μl at 6 months, 39.04±1.95 μl at 12 months and 41.04±3.05 μl at 18 months). Collectively, these data suggest that OGN regulates cardiac fibrosis during pressure overload and is therefore important in the remodeling response to hypertension and aging.

Emerging evidence indicates that inflammatory activation is a crucial pathway in disease progression during HF. The co-stimulatory dyad CD40-CD40L is involved in pro-inflammatory activation of a wide range of immune and non-immune cells and plasma levels of soluble CD40L in HF patients are raised. Here we investigated whether CD40-CD40L interactions attenuate cardiac hypertrophy in hypertensive heart disease. CD40 knockout and wild type C57Bl6-J mice were treated with angiotensin II (AngII, 2.5 mg/kg/day) for 4 weeks. AngII-induced hypertension led to a significant increase in cardiac mass in Wt mice (+31%, P

Rationale: Acute viral myocarditis (VM) is one of the leading causes of heart failure in young adults, lacking specific therapy. Osteonection (SPARC) is a non-structural matrix protein modulating cell-matrix interaction in the heart, and has been implicated in cardiac remodeling. Whether SPARC may regulate cardiac inflammation and necrosis in VM through its matri-cellular function is unknown. Methods and results: To address the implication of SPARC in VM, a human coxsackie B3 (CVB3) murine model of VM was performed in SPARC-null and WT animals. SPARC transcript- and protein levels significantly increased by 100% percent 1 week after CVB3 injection compared to control injection. Furthermore, absence of SPARC increased the mortality (4 out of 6 in null vs. 3 out of 7 in WT at 15 days), which was associated with increased cardiac inflammation and necrosis (9% ± 3.03 in KO vs. 1.3% ± 0.24 in WT). Concordantly, Adenoviral SPARC (adSPARC) over-expression, prior to CVB3 injection, improved the survival in both SPARC-null and WT mice, and reduced cardiac inflammation and necrosis by 55 percent in the SPARC null mice. Most importantly, adSPARC over-expression in WT animals resulted in improved cardiac function after viral injury as compared to control vector (fractional shortening was 28,2% ± 0,6 vs. 21,2% ± 0,8 respectively). Intra-vital microscopy of the cremaster muscle allowed us to study leukocyte behavior and permeability in vivo. This demonstrated significantly increased permeability with 450% and decreased leukocyte velocities in SPARC null mice upon TNF-α stimulation, in line with increased leukocyte extravasation. Moreover we performed in vivo measurement of the endothelial glycocalyx, a gel like structure at the luminal side of endothelial cells which is important in endothelial barrier properties. We found glycocalyx quality to be significantly diminished in SPARC null mice. VCAM-1 was further identified as a possible mediator of proper leukocyte recruitment through increased SPARC, since VCAM-1 staining clearly co-localized with SPARC at the endothelial cell level. Conclusion: Here, we demonstrated a protective role of SPARC in VM by improving cardiac function and reducing cardiac inflammation, necrosis and overall mortality. This protective role can be attributed to SPARC by improving endothelial barrier properties and thereby prohibiting exaggerated leukocyte recruitment. Clinical Relevance: As therapy for acute myocarditis is lacking and mainly supportive, we believe SPARC may be a novel target for the treatment of VM.

Background: Viral myocarditis is an important cause of heart failure and sudden cardiac death in young adults. Our previous studies have shown that many microRNAs are differentially expressed in the heart during viral myocarditis, including microRNA-221 and -222. Therefore we aimed at studying the biological role of these miRs during viral myocarditis in mice, induced by human Coxsackie B3 virus. Materials and methods: We measured cardiac microRNA levels during VM with microarrays and qRT-PCR. Wild type C3H mice were intraperitoneally infected with CVB3. Mice were sacrificed at day 4 and day 7 post infection. The hearts, and other organs, were weighted and collected for histological analyses, immunoblotting and qRT-PCR. In vitro experiments were performed in neonatal rat cardiomyocytes (nRCMs). Results: Levels of miR-221 and miR-222 were significantly elevated during acute viral myocarditis in mice, and depressed in biopsies from viral myocarditis patients. In situ hybridization and in vitro experiments showed that both miRs are expressed by cardiomyocytes and cardiac endothelial cells, and are up regulated in neonatal rat cardiomyocytes (nRCMs) in response to enteroviral infection. In vivo inhibition of miR-221 and miRNA-222 by systemically delivered antagomirs (day +1, +3 and +6) aggravated cardiac injury and inflammation at day 7 post-infection, related to a threefold increase of viral loads at day +7 as compared to scrambled antagomiRs. Likewise, inhibition of miR-221 and -222 significantly increased the number of viral particles in nRCMs, whereas overexpression of miR-221 and -222 in nRCMs inhibited enteroviral replication by 30 percent at 48h post-infection. We identified and confirmed a set of conserved miR-221/-222 targets, including ETS1 and ETS2, the chemokine CXCL12, the type I interferon inhibitor IRF2, the T cell development factor TOX and the anti-apoptotic proteins BCL2L11 and BMF. Furthermore, the Coxsackie and adenovirus receptor (CXADR), up regulated in biopsies of patients with dilated cardiomyopathy, is also targeted by miR-221/-222 in mice and rats. De-repression of CXADR upon inhibition of miR-221/-222 may explain the higher viral load observed in vivo and in vitro. Higher CXADR levels contribute to increased viral entry in the cardiomyocyte. Conclusions: Systemic inhibition of miR-221 and miR-222 increases initial cardiac viral loads and resulting inflammation and necrosis during VM in mice. Both targets involved in increased viral replication and enhanced inflammation with inhibition of the miR-221/-222 family are involved. This miR cluster thus represents a master switch in VM.

Background Viral myocarditis can severely damage the myocardium through excessive infiltration of immune cells. Osteoglycin (OGN) is part of the small leucine-rich repeat proteoglycan (SLRP) family. SLRP’s may affect inflammatory and fibrotic processes, but the implication of OGN in cardiac inflammation and the resulting injury upon viral myocarditis is unknown. Methods and results This study uncovered a previously unidentified 72-kDa variant of OGN that is predominant in cardiac human and mouse samples of viral myocarditis. Its absence in mice significantly decreased cardiac inflammation and injury in Coxsackievirus-B3-induced myocarditis. It also delayed mortality in lipopolysaccharide-induced endotoxemia going along with a reduced systemic production of pro-inflammatory cytokines. This 72-kDa OGN is expressed in the cell membrane of circulating and resident cardiac macrophages and neutrophils. Co-immunoprecipitation and OGN siRNA experiments revealed that this 72-kDa variant activates the toll-like receptor-4 (TLR4) with a concomitant increase in IL-6, TNF-α, IL-1β, and IL-12 expression. This immune cell activation by OGN occurred via MyD88 and increased phosphorylation of c-jun. Finally, the 72-kDa chondroitin sulfate is the result of O-linked glycosylation of the 32-kDa protein core of OGN. In contrast, the 34-kDa dermatan sulfate-OGN, involved in collagen cross linking, was also the result of O-linked glycosylation. Conclusion The current study discovered a novel 72-kDa chondroitin sulfate-OGN that is specific for innate immune cells. This variant is able to bind and activate TLR4. The absence of OGN decreases cytokine production by both circulating and cardiac leukocytes upon (systemic) LPS exposure, and reduces cardiac inflammation and injury in viral myocarditis. Electronic supplementary material The online version of this article (doi:10.1007/s00018-016-2423-7) contains supplementary material, which is available to authorized users.