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Abstract Aims We aim to characterize the clinical and proteomic profiles of patients at risk of developing heart failure (HF), with and without coronary artery disease (CAD) or prior myocardial infarction (MI). Methods and results HOMAGE evaluated the effect of spironolactone on plasma and serum markers of fibrosis over 9 months of follow‐up in participants with (or at risk of having) CAD, and raised natriuretic peptides. In this post hoc analysis, patients were classified as (i) neither CAD nor MI; (ii) CAD; or (iii) MI. Proteomic between‐group differences were evaluated through logistic regression and narrowed using backward stepwise selection and bootstrapping. Among the 527 participants, 28% had neither CAD or MI, 31% had CAD, and 41% had prior MI. Compared with people with neither CAD nor MI, those with CAD had higher baseline plasma concentrations of matrix metalloproteinase‐7 (MMP‐7), galectin‐4 (GAL4), plasminogen activator inhibitor 1 (PAI‐1), and lower plasma peptidoglycan recognition protein 1 (PGLYRP1), whilst those with a history of MI had higher plasma MMP‐7, neurotrophin‐3 (NT3), pulmonary surfactant‐associated protein D (PSPD), and lower plasma tumour necrosis factor‐related activation‐induced cytokine (TRANCE). Proteomic signatures were similar for patients with CAD or prior MI. Treatment with spironolactone was associated with an increase of MMP7, NT3, and PGLYRP1 at 9 months. Conclusions In patients at risk of developing HF, those with CAD or MI had a different proteomic profile regarding inflammatory, immunological, and collagen catabolic processes.

Peripheral artery disease (PAD), a significant health burden worldwide, affects lower extremities due to atherosclerosis in peripheral vessels. Although the mechanisms of PAD have been well studied, the molecular milieu of the plaques localized within peripheral arteries are not well understood. Thus, to identify PAD-lesion-specific gene expression profiles precluding genetic, environmental, and dietary biases, we studied the transcriptomic profile of nine plaque tissues normalized to non-plaque tissues from the same donors. A total of 296 upregulated genes, 274 downregulated genes, and 186 non-coding RNAs were identified. STAG1, SPCC3, FOXQ1, and E2F3 were key downregulated genes, and CD93 was the top upregulated gene. Autophagosome assembly, cellular response to UV, cytoskeletal organization, TCR signaling, and phosphatase activity were the key dysregulated pathways identified. Telomerase regulation and autophagy were identified as novel interacting pathways using network analysis. The plaque tissue was predominantly composed of immune cells and dedifferentiated cell populations indicated by cell-specific marker-imputed gene expression analysis. This study identifies novel genes, non-coding RNAs, associated regulatory pathways, and the cell composition of the plaque tissue in PAD patients. The autophagy and immunoregulatory genes may drive novel mechanisms, resulting in atheroma. These novel interacting networks and genes have potential for PAD-specific therapeutic applications.

Abstract Allele-specific expression (ASE) analysis detects the relative abundance of alleles at heterozygous loci as a proxy for cis-regulatory variation, which affects the personal transcriptome and proteome. This study describes the development and application of an ASE analysis pipeline on a unique cohort of 87 well phenotyped and RNA sequenced patients from the Maastricht Cardiomyopathy Registry with dilated cardiomyopathy (DCM), a complex genetic disorder with a remaining gap in explained heritability. Regulatory processes for which ASE is a proxy might explain this gap. We found an overrepresentation of known DCM-associated genes among the significant results across the cohort. In addition, we were able to find genes of interest that have not been associated with DCM through conventional methods such as genome-wide association or differential gene expression studies. The pipeline offers RNA sequencing data processing, individual and population level ASE analyses as well as group comparisons and several intuitive visualizations such as Manhattan plots and protein–protein interaction networks. With this pipeline, we found evidence supporting the case that cis-regulatory variation contributes to the phenotypic heterogeneity of DCM. Additionally, our results highlight that ASE analysis offers an additional layer to conventional genomic and transcriptomic analyses for candidate gene identification and biological insight.

Abstract Aims In people at risk of heart failure (HF) enrolled in the Heart ‘OMics’ in AGEing (HOMAGE) trial, spironolactone reduced circulating markers of collagen synthesis, natriuretic peptides, and blood pressure and improved cardiac structure and function. In the present report, we explored factors associated with dyskalaemia. Methods and results The HOMAGE trial was an open‐label study comparing spironolactone (up to 50 mg/day) versus standard care in people at risk for HF. After randomization, serum potassium was assessed at 1 and 9 months and was defined as low when ≤3.5 mmol/L (hypokalaemia) and high when ≥5.5 mmol/L (hyperkalaemia). Multivariable logistic regression models were constructed to identify clinical predictors of dyskalaemia. A total of 513 participants (median age 74 years, 75% men, median estimated glomerular filtration rate 71 mL/min/1.73 m2) had serum potassium available and were included in this analysis. At randomization, 88 had potassium 5.0 mmol/L. During follow‐up, on at least one occasion, a serum potassium 5.0 mmol/L was observed in 38 (8%) and >5.5 mmol/L in 5 (1.0%) participants. The median (percentile25−75) increase in serum potassium with spironolactone during the study was 0.23 (0.16; 0.29) mmol/L. Because of the low incidence of dyskalaemia, for regression analysis, hypokalaemia and hyperkalaemia thresholds were set at 5.0 mmol/L, respectively. The occurrence of a serum potassium > 5.0 mmol/L during follow‐up was positively associated with the presence of diabetes mellitus {odds ratio [OR]: 1.21 [95% confidence interval (CI) 2.14; 3.79]} and randomization to spironolactone (OR: 2.83 [95% CI 1.49; 5.37]). Conversely, the occurrence of a potassium concentration

The role of ADAMTSL3 in heart pathology and cardiac disease is investigated with Adamtsl3-deficient mice using CRISPR-Cas9 gene editing.

BackgroundAnthracycline-induced cardiotoxicity is a well-known serious clinical entity. However, detailed mechanistic insights on how short-term administration leads to late and long-lasting cardiotoxicity, are still largely undiscovered. We hypothesize that chemotherapy provokes a memory effect at the level of epigenomic DNA modifications which subsequently lead to cardiotoxicity even years after cessation of chemotherapy.MethodsWe explored the temporal evolution of epigenetic modifiers in early and late cardiotoxicity due to anthracyclines by means of RNA-sequencing of human endomyocardial left ventricular biopsies and mass spectrometry of genomic DNA. Based on these findings, validation of differentially regulated genes was obtained by performing RT-qPCR. Finally, a proof-of-concept in vitro mechanistic study was performed to dissect some of the mechanistic aspects of epigenetic memory in anthracycline-induced cardiotoxicity.ResultsCorrelation of gene expression between late and early onset cardiotoxicity revealed an R2 value of 0.98, demonstrating a total of 369 differentially expressed genes (DEGs, FDR

Abstract Viral myocarditis (VM) is an important cause of heart failure (HF) in children and adults. However, the molecular determinants involved in cardiac inflammation and cardiomyocyte necrosis remain poorly characterized, and cardioprotective molecules are currently missing. Here, we applied an in vivo method based on the functional selection (FunSel) of cardioprotective factors using AAV vectors for the unbiased identification of novel immunomodulatory molecules in a Coxsackievirus B3 (CVB3)-induced myocarditis mouse model. Two consecutive rounds of in vivo FunSel using an expression library of 60 cytokines were sufficient to identify five cardioprotective factors (IL9, IL3, IL4, IL13, IL15). The screening also revealed three cytokines (IL18, IL17b, and CCL11) that were counter-selected and likely to exert a detrimental effect. The pooled overexpression of the five most enriched cytokines using AAV9 vectors decreased inflammation and reduced cardiac dilatation, persisting at 1 month after treatment. Individual overexpression of IL9, the top ranking in our functional selection, markedly reduced cardiac inflammation and injury, concomitant with an increase of anti-inflammatory Th2-cells and a reduction of pro-inflammatory Th17- and Th22-cells at 14 days post-infection. AAV9-mediated FunSel cardiac screening identified IL9 and other four cytokines (IL3, IL4, IL13, and IL15) as cardioprotective factors in CVB3-induced VM in mice.

Abstract Aims Heart failure (HF) is common in both men and women, yet disease pathophysiology, presentation, and progression differ between sexes. Studies addressing whether biomarkers predict new onset HF sex‐specifically are scarce. This study therefore aims to test the sex‐specificity of 252 protein biomarkers for new‐onset HF. Methods and results A matched case–control design in patients selected from cohorts within the HOMAGE consortium was used. Cases (new‐onset HF, n = 562) and controls (n = 780) were matched for cohort (PREDICTOR, HEALTH‐ABC, & PROSPER), follow‐up time (defined as time from entry to incident HF), and age. Incident HF was defined as first hospitalization for HF. Targeted plasma proteins (n = 252) were measured using Proximity Extension Assay technology from O‐link. To look for sex differences for new onset HF, we adjusted for cohort, age, and baseline clinical parameters. At baseline, women had a biomarker profile reflecting activated metabolism and immune responses. However, none of the biomarkers had a significant interaction with sex in predicting new onset HF, but four biomarkers had a trend towards sex‐specificity (P

Recovered COVID-19 patients often display cardiac dysfunction, even after a mild infection. Most current histological results come from patients that are hospitalized and therefore represent more severe outcomes than most COVID-19 patients face. To overcome this limitation, we investigated the cardiac effects of SARS-CoV-2 infection in a hamster model. SARS-CoV-2 infected hamsters developed diastolic dysfunction after recovering from COVID-19. Histologically, increased cardiomyocyte size was present at the peak of viral load and remained at all time points investigated. As this increase is too rapid for hypertrophic remodeling, we found instead that the heart was oedemic. Moreover, cardiomyocyte swelling is associated with the presence of ischemia. Fibrin-rich microthrombi and pericyte loss were observed at the peak of viral load, resulting in increased HIF1α in cardiomyocytes. Surprisingly, SARS-CoV-2 infection inhibited the translocation of HIF1α to the nucleus both in hamster hearts, in cultured cardiomyocytes, as well as in an epithelial cell line. We propose that the observed diastolic dysfunction is the consequence of cardiac oedema, downstream of microvascular cardiac ischemia. Additionally, our data suggest that inhibition of HIF1α translocation could contribute to an exaggerated response upon SARS-CoV-2 infection.

Abstract 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 human 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 that can be prevented by NOTCH signaling inhibitors. Moreover, valproic acid pre-treated cdMiPs injected in dystrophic muscles increase physical strength and ameliorate the functional performances of transplanted mice. Taken together, these results constitute a novel approach to generate mesodermal progenitors with enhanced myogenic potential using clinically approved reagents.

Background: Chagas disease is an endemic protozoan disease with high prevalence in Latin America. Of those infected, 20–30% will develop chronic Chagas cardiomyopathy (CCC) however, prediction using existing clinical criteria remains poor. In this study, we investigated the utility of left ventricular (LV) echocardiographic speckle-tracking global longitudinal strain (GLS) for early detection of CCC. Methods and results: 139 asymptomatic T. cruzi seropositive subjects with normal heart size and normal LV ejection fraction (EF) (stage A or B) were enrolled in this prospective observational study and underwent paired echocardiograms at baseline and 1-year follow-up. Progressors were participants classified as stage C or D at follow-up due to development of symptoms of heart failure, cardiomegaly, or decrease in LVEF. LV GLS was calculated as the average peak systolic strain of 16 LV segments. Measurements were compared between participants who progressed and did not progress by two-sample t-test, and the odds of progression assessed by multivariable logistic regression. Of the 139 participants, 69.8% were female, mean age 55.8 ± 12.5 years, with 12 (8.6%) progressing to Stage C or D at follow-up. Progressors tended to be older, male, with wider QRS duration. LV GLS was −19.0% in progressors vs. –22.4% in non-progressors at baseline, with 71% higher odds of progression per +1% of GLS (adjusted OR 1.71, 95% CI 1.20–2.44, p = 0.003). Conclusion: Baseline LV GLS in participants with CCC stage A or B was predictive of progression within 1-year and may guide timing of clinical follow-up and promote early detection or treatment.

Circulating biomarkers reflecting cardiac inflammation are needed to improve the diagnostics and guide the treatment of heart failure patients. The cardiac production and shedding of the transmembrane proteoglycan syndecan-4 is upregulated by innate immunity signaling pathways. Here, we investigated the potential of syndecan-4 as a blood biomarker of cardiac inflammation. Serum syndecan-4 was measured in patients with (i) non-ischemic, non-valvular dilated cardiomyopathy (DCM), with (n = 71) or without (n = 318) chronic inflammation; (ii) acute myocarditis (n = 15), acute pericarditis (n = 3) or acute perimyocarditis (23) and (iii) acute myocardial infarction (MI) at day 0, 3 and 30 (n = 119). Syndecan-4 was investigated in cultured cardiac myocytes and fibroblasts (n = 6–12) treated with the pro-inflammatory cytokines interleukin (IL)-1β and its inhibitor IL-1 receptor antagonist (IL-1Ra), or tumor necrosis factor (TNF)α and its specific inhibitor infliximab, an antibody used in treatment of autoimmune diseases. The levels of serum syndecan-4 were comparable in all subgroups of patients with chronic or acute cardiomyopathy, independent of inflammation. Post-MI, syndecan-4 levels were increased at day 3 and 30 vs. day 0. IL-1Ra attenuated IL-1β-induced syndecan-4 production and shedding in vitro, while infliximab had no effect. In conclusion, syndecan-4 shedding from cardiac myocytes and fibroblasts was attenuated by immunomodulatory therapy. Although its circulating levels were increased post-MI, syndecan-4 did not reflect cardiac inflammatory status in patients with heart disease.

Abstract Background Congenital disorders of glycosylation (CDG) are a group of metabolic disorders well known to be associated with developmental delay and central nervous system anomalies. The most common CDG is caused by pathogenic variants in the phosphomannomutase 2 gene (PMM2), which impairs one of the first steps of N‐glycosylation and affects multiple organ systems. Cardiac involvement can include pericardial effusion, cardiomyopathy, and arrhythmia, while an association with cardiovascular congenital anomalies is not well studied. Case summary We report a 6‐year‐old individual who initially presented with inverted nipples, developmental delay, and failure to thrive at 3 months of age. At 4 months, due to feeding problems, swallowing exam and echocardiography were performed which revealed a vascular ring anomaly based on a right aortic arch and aberrant left subclavian artery. Subsequent whole exome gene sequencing revealed two pathogenic PMM2‐CDG variants (E139K/R141H) and no known pathogenic mutations related to congenital heart defect (CHD). Discussion This is the first report of vascular ring anomaly in a patient with PMM2‐CDG. We conducted a literature review of PMM2‐CDG patients with reported CHD. Of the 14 patients with PMM2‐CDG and cardiac malformation, the most common CHD's were tetralogy of Fallot, patent ductus arteriosus, and truncus arteriosus. The potential important link between CDG and CHD is stressed and discussed. Furthermore, the importance of multidisciplinary care for CDG patients including early referral to pediatric cardiologists is highlighted.

Abstract Aims Chronic non‐viral myocarditis, also called inflammatory cardiomyopathy, can be treated with immune suppression on tops of optimal medical therapy (OMT) for heart failure, using a combination of prednisolone and azathioprine (IPA). However, there has been inconsistency in the effects of immunosuppression treatment. This meta‐analysis is the first to evaluate all available data of the effect of this treatment on left ventricular ejection fraction (LVEF) and the combined clinical endpoint of cardiovascular mortality and/or heart transplantation‐free survival. Methods and results All trials with using IPA vs. OMT in this syndrome were searched using OVID Medline and ClinicalTrials.gov, following the PRISMA guidelines. Missing data were retrieved after contacting the corresponding authors. All data was reviewed and analysed using and standard meta‐analysis methods. A random effect model was used to pool the effect sizes. A total of four trials (three randomised controlled trials and one propensity‐matched retrospective registry) including 369 patients were identified. IPA on top of OMT did not improve LVEF [mean difference 9.9% (95% confidence interval −1.8, 21.7)] with significant heterogeneity. When we limited our pooled estimate to the published studies only, significant LVEF improvement by IPA was observed [14% (1.4, 26.6)]. No cardiovascular mortality benefit was observed with the intervention [risk ratio 0.34 (0.08, 1.51)]. Conclusions At the moment, there is insufficient evidence supporting functional and prognostic benefits of IPA added to OMT in virus negative inflammatory positive cardiomyopathy. Further adequate‐powered well‐designed prospective RCTs should be warranted to explore the potential effects of adding immunosuppressive therapy to OMT.

Host defense peptides represent an important component of innate immunity. In this work, we report the anticancer properties of a panel of hyper-charged wholly cationic antimicrobial dodecapeptides (CAPs) containing multiple canonical forms of lysine and arginine residues. These CAPs displayed excellent bactericidal activities against a broad range of pathogenic bacteria by dissipating the cytoplasmic membrane potential. Specifically, we identified two CAPs, named HC3 and HC5, that effectively killed a significant number of retinoblastoma (WERI-Rb1) cells (p ≤ 0.01). These two CAPs caused the shrinkage of WERI-Rb1 tumor spheroids (p ≤ 0.01), induced intrinsic apoptosis in WERI-Rb1 cells via activation of caspase 9 and caspase 3, cleaved the PARP protein, and triggered off the phosphorylation of p53 and γH2A.X. Combining HC3 or HC5 with the standard chemotherapeutic drug topotecan showed synergistic anti-cancer activities. Overall, these results suggest that HC3 and HC5 can be exploited as potential therapeutic agents in retinoblastoma as monotherapy or as adjunctive therapy to enhance the effectiveness of currently used treatment modalities.

Mutations in the RB1 locus leading to a loss of functional Rb protein cause intraocular tumors, which uniquely affect children worldwide. These tumors demonstrate rapid proliferation, which has recently been shown to be associated with an altered metabolic signature. We found that retinoblastoma tumors and in-vitro models lack Hexokinase 1 (HK1) and exhibit elevated fatty acid oxidation. We show that ectopic expression of RB1 induces HK1 protein in Rb null cells, and both RB1 and HK1 can mediate a metabolic switch from OXPHOS to glycolysis with increased pyruvate levels, reduced ATP production and reduced mitochondrial mass. Further, cells lacking Rb or HK1 can flexibly utilize glutamine and fatty acids to enhance oxidative phosphorylation-dependent ATP generation, as revealed by metabolic and biochemical assays. Thus, loss of Rb and HK1 in retinoblastoma reprograms tumor metabolic circuits to enhance the glucose-independent TCA (tricarboxylic acid) cycle and the intermediate NAD+/NADH ratios, with a subsequent increase in fatty-acid derived L-carnitine to enhance mitochondrial OXPHOS for ATP production instead of glycolysis dependence. We also demonstrate that modulation of the Rb-regulated transcription factor E2F2 does not result in any of these metabolic perturbations. In conclusion, we demonstrate RB1 or HK1 as critical regulators of the cellular bioenergetic profile and identify the altered tumor metabolism as a potential therapeutic target for cancers lacking functional Rb protein.

Summary: The mechanism of action of empagliflozin in heart failure with reduced ejection fraction (HFrEF) was deciphered using deep learning in silico analyses together with in vivo validation. The most robust mechanism of action involved the sodium-hydrogen exchanger (NHE)-1 co-transporter with 94.7% accuracy, which was similar for diabetics and nondiabetics. Notably, direct NHE1 blockade by empagliflozin ameliorated cardiomyocyte cell death by restoring expression of X-linked inhibitor of apoptosis (XIAP) and baculoviral IAP repeat-containing protein 5 (BIRC5). These results were independent of diabetes mellitus comorbidity, suggesting that empagliflozin may emerge as a new treatment in HFrEF. Key Words: empagliflozin, heart failure, machine learning

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

Retinoblastoma (Rb) is a pediatric intraocular malignancy that is proposed to originate from maturing cone cell precursors in the developing retina. The molecular mechanisms underlying the biological and clinical behaviors are important to understand in order to improve the management of advanced-stage tumors. While the genetic causes of Rb are known, an integrated understanding of the gene expression and metabolic processes in tumors of human eyes is deficient. By integrating transcriptomic profiling from tumor tissues and metabolomics from tumorous eye vitreous humor samples (with healthy, age-matched pediatric retinae and vitreous samples as controls), we uncover unique functional associations between genes and metabolites. We found distinct gene expression patterns between clinically advanced and non-advanced Rb. Global metabolomic analysis of the vitreous humor of the same Rb eyes revealed distinctly altered metabolites, indicating how tumor metabolism has diverged from healthy pediatric retina. Several key enzymes that are related to cellular energy production, such as hexokinase 1, were found to be reduced in a manner corresponding to altered metabolites; notably, a reduction in pyruvate levels. Similarly, E2F2 was the most significantly elevated E2F family member in our cohort that is part of the cell cycle regulatory circuit. Ectopic expression of the wild-type RB1 gene in the Rb-null Y79 and WERI-Rb1 cells rescued hexokinase 1 expression, while E2F2 levels were repressed. In an additional set of Rb tumor samples and pediatric healthy controls, we further validated differences in the expression of HK1 and E2F2. Through an integrated omics analysis of the transcriptomics and metabolomics of Rb, we uncovered a significantly altered tumor-specific metabolic circuit that reduces its dependence on glycolytic pathways and is governed by Rb1 and HK1.

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 at 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 increases 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.

Myocarditis is an inflammatory disease of the heart with viral infections being the most common aetiology. Its complex biology remains poorly understood and its clinical management is one of the most challenging in the field of cardiology. Toll-like receptors (TLRs), a family of evolutionarily conserved pattern recognition receptors, are increasingly known to be implicated in the pathophysiology of viral myocarditis. Their central role in innate and adaptive immune responses, and in the inflammatory reaction that ensues, indeed makes them prime candidates to profoundly affect every stage of the disease process. This review describes the pathogenesis and pathophysiology of viral myocarditis, and scrutinises the role of TLRs in every phase. We conclude with directions for future research in this field.

Heart failure (HF) is the most rapidly growing cardiovascular health burden worldwide. HF can be classified into three groups based on the percentage of the ejection fraction (EF): heart failure with reduced EF (HFrEF), heart failure with mid-range—also called mildly reduced EF— (HFmrEF), and heart failure with preserved ejection fraction (HFpEF). HFmrEF can progress into either HFrEF or HFpEF, but its phenotype is dominated by coronary artery disease, as in HFrEF. HFrEF and HFpEF present with differences in both the development and progression of the disease secondary to changes at the cellular and molecular level. While recent medical advances have resulted in efficient and specific treatments for HFrEF, these treatments lack efficacy for HFpEF management. These differential response rates, coupled to increasing rates of HF, highlight the significant need to understand the unique pathogenesis of HFrEF and HFpEF. In this review, we summarize the differences in pathological development of HFrEF and HFpEF, focussing on disease-specific aspects of inflammation and endothelial function, cardiomyocyte hypertrophy and death, alterations in the giant spring titin, and fibrosis. We highlight the areas of difference between the two diseases with the aim of guiding research efforts for novel therapeutics in HFrEF and HFpEF.

Cardiovascular disease (CVD) is one of the biggest threats to public health worldwide. Identifying key genetic contributors to CVD enables clinicians to assess the most effective treatment course and prognosis, as well as potentially inform family members. This often involves either whole exome sequencing (WES) or targeted panel analysis of known pathogenic genes. In the future, tailored or personalized therapeutic strategies may be implemented, such as gene therapy. With the recent revolution in deep sequencing technologies, we know that up to 90% of the human genome is transcribed, despite only 2% of the 6 billion DNA bases coding for proteins. The long non-coding RNA (lncRNA) “genes” make up an important and significant fraction of this “dark matter” of the genome. We highlight how, despite lncRNA genes exceeding that of classical protein-coding genes by number, the “non-coding” human genome is neglected when looking for genetic components of disease. WES platforms and pathogenic gene panels still do not cover even characterized lncRNA genes that are functionally involved in the pathophysiology of CVD. We suggest that the importance of lncRNAs in disease causation and progression be taken as seriously as that of pathogenic protein variants and mutations, and that this is maybe a new area of attention for clinical geneticists.

Cardiovascular disease (CVD) remains the leading cause of death worldwide and, despite continuous advances, better diagnostic and prognostic tools, as well as therapy, are needed. The human transcriptome, which is the set of all RNA produced in a cell, is much more complex than previously thought and the lack of dialogue between researchers and industrials and consensus on guidelines to generate data make it harder to compare and reproduce results. This European Cooperation in Science and Technology (COST) Action aims to accelerate the understanding of transcriptomics in CVD and further the translation of experimental data into usable applications to improve personalized medicine in this field by creating an interdisciplinary network. It aims to provide opportunities for collaboration between stakeholders from complementary backgrounds, allowing the functions of different RNAs and their interactions to be more rapidly deciphered in the cardiovascular context for translation into the clinic, thus fostering personalized medicine and meeting a current public health challenge. Thus, this Action will advance studies on cardiovascular transcriptomics, generate innovative projects, and consolidate the leadership of European research groups in the field. COST (European Cooperation in Science and Technology) is a funding organization for research and innovation networks (www.cost.eu).

Abstract Myocarditis, often triggered by viral infection, may lead to heart auto‐immunity and dilated cardiomyopathy. What determines the switch between disease resolution and progression is however incompletely understood. We show that pharmacological inhibition of STAT3, the main mediator of IL‐6 signalling and of Th17‐cell differentiation, protects mice from the development of Experimental Auto‐immune Myocarditis reducing liver production of the complement component C3, and can act therapeutically when administered at disease peak. Further, we demonstrate that STAT3 is sufficient when constitutively active for triggering the onset of immune‐mediated myocarditis, involving enhanced complement C3 production and IL‐6 signalling amplification in the liver. Disease development can be prevented by C3 depletion and IL‐6 receptor neutralization. This appears to be relevant to disease pathogenesis in humans, since acute myocarditis patients display significantly elevated circulating IL‐6 and C3 levels and activated heart STAT3. Thus, aberrant IL‐6/STAT3‐mediated induction of liver acute phase response genes including C3, which occurs as a consequence of pre‐existing inflammatory conditions, might represent an important factor determining the degree of myocarditis and its clinical outcome.

Long non-coding RNAs (lncRNAs) are a class of RNA molecules with diverse regulatory functions during embryonic development, normal life, and disease in higher organisms. However, research on the role of lncRNAs in cardiovascular diseases and in particular heart failure is still in its infancy. The exceptionally well conserved nuclear lncRNA Metastasis associated in lung adenocarcinoma transcript 1 (Malat-1) is a regulator of mRNA splicing and highly expressed in the heart. Malat-1 modulates hypoxia-induced vessel growth, activates ERK/MAPK signaling, and scavenges the anti-hypertrophic microRNA-133. We therefore hypothesized that Malat-1 may act as regulator of cardiac hypertrophy and failure during cardiac pressure overload induced by thoracic aortic constriction (TAC) in mice.Absence of Malat-1 did not affect cardiac hypertrophy upon pressure overload: Heart weight to tibia length ratio significantly increased in WT mice (sham: 5.78±0.55, TAC 9.79±1.82 g/mm; p

Von Willebrand A domain Related Protein (WARP), is a recently identified extracellular matrix protein. Based upon its involvement in matrix biology and its expression in the heart, we hypothesized that WARP regulates cardiac remodeling processes in the post-infarct healing process.In the mouse model of myocardial infarction (MI), WARP expression increased in the infarcted area 3-days post-MI. In the healthy myocardium WARP localized with perlecan in the basement membrane, which was disrupted upon injury. In vitro studies showed high expression of WARP by cardiac fibroblasts, which further increases upon TGFβ stimulation. Furthermore, WARP expression correlated with aSMA and COL1 expression, markers of fibroblast to myofibroblast transition, in vivo and in vitro. Finally, WARP knockdown in vitro affected extra- and intracellular basic fibroblast growth factor production in myofibroblasts. To investigate the function for WARP in infarction healing, we performed an MI study in WARP knockout (KO) mice backcrossed more than 10 times on an Australian C57Bl/6-J background and bred in-house, and compared to wild type (WT) mice of the same C57Bl/6-J strain but of commercial European origin. WARP KO mice showed no mortality after MI, whereas 40% of the WT mice died due to cardiac rupture. However, when WARP KO mice were backcrossed on the European C57Bl/6-J background and bred heterozygous in-house, the previously seen protective effect in the WARP KO mice after MI was lost. Importantly, comparison of the cardiac response post-MI in WT mice bred heterozygous in-house versus commercially purchased WT mice revealed differences in cardiac rupture.These data demonstrate a redundant role for WARP in the wound healing process after MI but demonstrate that the continental/breeding/housing origin of mice of the same C57Bl6-J strain is critical in determining the susceptibility to cardiac rupture and stress the importance of using the correct littermate controls.

MicroRNAs (miRNAs) are a class of non-coding RNAs of ∼22 nucleotides in length, and constitute a novel class of gene regulators by imperfect base-pairing to the 3'UTR of protein encoding messenger RNAs. Growing evidence indicates that miRNAs are implicated in several pathological processes in myocardial disease. The past years, we have witnessed several profiling attempts using high-density oligonucleotide array-based approaches to identify the complete miRNA content (miRNOME) in the healthy and diseased mammalian heart. These efforts have demonstrated that the failing heart displays differential expression of several dozens of miRNAs. While the total number of experimentally validated human miRNAs is roughly two thousand, the number of expressed miRNAs in the human myocardium remains elusive. Our objective was to perform an unbiased assay to identify the miRNOME of the human heart, both under physiological and pathophysiological conditions. We used deep sequencing and bioinformatics to annotate and quantify microRNA expression in healthy and diseased human heart (heart failure secondary to hypertrophic or dilated cardiomyopathy). Our results indicate that the human heart expresses >800 miRNAs, the majority of which not being annotated nor described so far and some of which being unique to primate species. Furthermore, >250 miRNAs show differential and etiology-dependent expression in human dilated cardiomyopathy (DCM) or hypertrophic cardiomyopathy (HCM). The human cardiac miRNOME still possesses a large number of miRNAs that remain virtually unexplored. The current study provides a starting point for a more comprehensive understanding of the role of miRNAs in regulating human heart disease.

microRNA-155 (miR155) is a central regulator of immune responses that is induced by inflammatory mediators. Although miR155 is considered to be a pro-inflammatory microRNA, in vitro reports show anti-inflammatory effects in lipid-loaded cells. In this study we examined the role of miR155 in atherosclerosis in vivo using bone marrow transplantation from miR155 deficient or wildtype mice to hyperlipidemic mice. Hematopoietic deficiency of miR155 enhanced atherosclerotic plaque development and decreased plaque stability, as evidenced by increased myeloid inflammatory cell recruitment to the plaque. The increased inflammatory state was mirrored by a decrease in circulating CD4(+)CD25(+)FoxP3(+) regulatory T cells, and an increase in granulocytes (CD11b(+)Ly6G(+)) in blood of miR155(-/-) transplanted mice. Moreover, we show for the first time a crucial role of miR155 in monocyte subset differentiation, since hematopoietic deficiency of miR155 increases the 'inflammatory' monocyte subset (CD11b(+)Ly6G(-)Ly6C(hi)) and reduces 'resident' monocytes (CD11b(+)Ly6G(-)Ly6C(low)) in the circulation. Furthermore, cytokine production by resident peritoneal macrophages of miR155(-/-) transplanted hyperlipidemic mice was skewed towards a more pro-inflammatory state since anti-inflammatory IL-10 production was reduced. In conclusion, in this hyperlipidemic mouse model miR155 acts as an anti-inflammatory, atheroprotective microRNA. Additionally, besides a known role in lymphoid cell development, we show a crucial role of miR155 in myeloid lineage differentiation.