Myocardial B cells have specific gene expression and predicted interactions in dilated cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy.

Introduction: Growing evidence from animal models indicates that the myocardium hosts a population of B cells that play a role in the development of cardiomyopathy. However, there is minimal data on human myocardial B cells in the context of cardiomyopathy. Methods: We integrated single-cell and single-nuclei datasets from 45 healthy human hearts, 70 hearts with dilated cardiomyopathy (DCM), and 8 hearts with arrhythmogenic right ventricular cardiomyopathy (ARVC). Interactions between B cells and other cell types were investigated using the CellChat Package. Differential gene expression analysis comparing B cells across conditions was performed using DESeq2. Pathway analysis was performed using Ingenuity, KEGG, and GO pathways analysis. Results: We identified 1,100 B cells, including naive B cells and plasma cells. Cells showed an extensive network of interactions within the healthy myocardium that included outgoing signaling to macrophages, T cells, endothelial cells, and pericytes, and incoming signaling from endothelial cells, pericytes, and fibroblasts. This niche relied on ECM-receptor, contact, and paracrine interactions; and changed significantly in the context of cardiomyopathy, displaying disease-specific features. Differential gene expression analysis showed that in the context of DCM both naive and plasma B cells upregulated several pathways related to immune activation, including upregulation of oxidative phosphorylation, upregulation of leukocyte extravasation, and, in naive B cells, antigen presentation. Discussion: The human myocardium contains naive B cells and plasma cells, integrated into a diverse and dynamic niche that has distinctive features in healthy, DCM, and ARVC. Naive myocardial-associated B cells likely contribute to the pathogenesis of human DCM.

Implementing a New Algorithm for Reinterpretation of Ambiguous Variants in Genetic Dilated Cardiomyopathy.

Dilated cardiomyopathy is a heterogeneous entity that leads to heart failure and malignant arrhythmias. Nearly 50% of cases are inherited; therefore, genetic analysis is crucial to unravel the cause and for the early identification of carriers at risk. A large number of variants remain classified as ambiguous, impeding an actionable clinical translation. Our goal was to perform a comprehensive update of variants previously classified with an ambiguous role, applying a new algorithm of already available tools. In a cohort of 65 cases diagnosed with dilated cardiomyopathy, a total of 125 genetic variants were classified as ambiguous. Our reanalysis resulted in the reclassification of 12% of variants from an unknown to likely benign or likely pathogenic role, due to improved population frequencies. For all the remaining ambiguous variants, we used our algorithm; 60.9% showed a potential but not confirmed deleterious role, and 24.5% showed a potential benign role. Periodically updating the population frequencies is a cheap and fast action, making it possible to clarify the role of ambiguous variants. Here, we perform a comprehensive reanalysis to help to clarify the role of most of ambiguous variants. Our specific algorithms facilitate genetic interpretation in dilated cardiomyopathy.

Gene-expression profiling of endomyocardial biopsies from dogs with dilated cardiomyopathy phenotype.

INTRODUCTION: The employment of advanced molecular biology technologies has expanded the diagnostic investigation of cardiomyopathies in dogs; these technologies have predominantly been performed on postmortem samples, although the recent use of endomyocardial biopsy in living dogs has enabled a better premortem diagnostic approach to study the myocardial injury. ANIMALS, MATERIALS, AND METHODS: Endomyocardial biopsies were collected in nine dogs with a dilated cardiomyopathy phenotype (DCM-p) and congestive heart failure and submitted to histologic examination, next-generation sequencing (NGS), and polymerase chain reaction analysis. Data from three healthy dogs (Fastq files) were retrieved from a previously approved study and used as a control group for ribonucleic acid sequencing. RESULTS: Histologic examination revealed endocardial fibrosis in six of nine dogs, whereas lymphocytic interstitial infiltrates were detected in two of nine dogs, and lymphoplasmacytic and macrophage infiltrates were detected in one of nine dogs. On polymerase chain reaction analysis, two dogs tested positive for canine parvovirus two and one dog for canine distemper virus. Gene-expression pathways involved in cellular energy metabolism (especially carbohydrates-insulin) and cardiac structural proteins were different in all DCM-p dogs compared to those in the control group. When dogs with lymphocytic interstitial infiltrates were compared to those in the control group, NGS analysis revealed the predominant role of genes related to inflammation and pathogen infection. CONCLUSIONS: Next-generation sequencing technology performed on in vivo endomyocardial biopsies has identified different molecular and genetic factors that could play a role in the development and/or progression of DCM-p in dogs.

Imagenetics for Precision Medicine in Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM) is a common heart muscle disorder of nonischemic etiology associated with heart failure development and the risk of malignant ventricular arrhythmias and sudden cardiac death. A tailored approach to risk stratification and prevention of sudden cardiac death is required in genetic DCM given its variable presentation and phenotypic severity. Currently, advances in cardiogenetics have shed light on disease mechanisms, the complex genetic architecture of DCM, polygenic contributors to disease susceptibility and the role of environmental triggers. Parallel advances in imaging have also enhanced disease recognition and the identification of the wide spectrum of phenotypes falling under the DCM umbrella. Genotype-phenotype associations have been also established for specific subtypes of DCM, such as DSP (desmoplakin) or FLNC (filamin-C) cardiomyopathy but overall, they remain elusive and not readily identifiable. Also, despite the accumulated knowledge on disease mechanisms, certain aspects remain still unclear, such as which patients with DCM are at risk for disease progression or remission after treatment. Imagenetics, that is, the combination of imaging and genetics, is expected to further advance research in the field and contribute to precision medicine in DCM management and treatment. In the present article, we review the existing literature in the field, summarize the established knowledge and emerging data on the value of genetics and imaging in establishing genotype-phenotype associations in DCM and in clinical decision making for DCM patients.

Role of arrhythmic phenotype in prognostic stratification and management of dilated cardiomyopathy.

AIMS: Dilated cardiomyopathy (DCM) with arrhythmic phenotype combines phenotypical aspects of DCM and predisposition to ventricular arrhythmias, typical of arrhythmogenic cardiomyopathy. The definition of DCM with arrhythmic phenotype is not universally accepted, leading to uncertainty in the identification of high-risk patients. This study aimed to assess the prognostic impact of arrhythmic phenotype in risk stratification and the correlation of arrhythmic markers with high-risk arrhythmogenic gene variants in DCM patients. METHODS AND RESULTS: In this multicentre study, DCM patients with available genetic testing were analysed. The following arrhythmic markers, present at baseline or within 1 year of enrolment, were tested: unexplained syncope, rapid non-sustained ventricular tachycardia (NSVT), ≥1000 premature ventricular contractions/24 h or ≥50 ventricular couplets/24 h. LMNA, FLNC, RBM20, and desmosomal pathogenic or likely pathogenic gene variants were considered high-risk arrhythmogenic genes. The study endpoint was a composite of sudden cardiac death and major ventricular arrhythmias (SCD/MVA). We studied 742 DCM patients (45 ± 14 years, 34% female, 410 [55%] with left ventricular ejection fraction [LVEF] <35%). During a median follow-up of 6 years (interquartile range 1.6-12.1), unexplained syncope and NSVT were the only arrhythmic markers associated with SCD/MVA, and the combination of the two markers carried a significant additive risk of SCD/MVA, incremental to LVEF and New York Heart Association class. The probability of identifying an arrhythmogenic genotype rose from 8% to 30% if both early syncope and NSVT were present. CONCLUSION: In DCM patients, the combination of early detected NSVT and unexplained syncope increases the risk of life-threatening arrhythmic outcomes and can aid the identification of carriers of malignant arrhythmogenic genotypes.

Missense variants in phospholamban and cardiac myosin binding protein identified in patients with a family history and clinical diagnosis of dilated cardiomyopathy.

As the genetic landscape of cardiomyopathies continues to expand, the identification of missense variants in disease-associated genes frequently leads to a classification of variant of uncertain significance (VUS). For the proper reclassification of such variants, functional characterization is an important contributor to the proper assessment of pathogenic potential. Several missense variants in the calcium transport regulatory protein phospholamban have been associated with dilated cardiomyopathy. However, >40 missense variants in this transmembrane peptide are currently known and most remain classified as VUS with little clinical information. Similarly, missense variants in cardiac myosin binding protein have been associated with hypertrophic cardiomyopathy. However, hundreds of variants are known and many have low penetrance and are often found in control populations. Herein, we focused on novel missense variants in phospholamban, an Ala15-Thr variant found in a 4-year-old female and a Pro21-Thr variant found in a 60-year-old female, both with a family history and clinical diagnosis of dilated cardiomyopathy. The patients also harbored a Val896-Met variant in cardiac myosin binding protein. The phospholamban variants caused defects in the function, phosphorylation, and dephosphorylation of this calcium transport regulatory peptide, and we classified these variants as potentially pathogenic. The variant in cardiac myosin binding protein alters the structure of the protein. While this variant has been classified as benign, it has the potential to be a low-risk susceptibility variant because of the structural change in cardiac myosin binding protein. Our studies provide new biochemical evidence for missense variants previously classified as benign or VUS.

LncRNA CHKB-DT Downregulation Enhances Dilated Cardiomyopathy Through ALDH2.

BACKGROUND: Human cardiac long noncoding RNA (lncRNA) profiles in patients with dilated cardiomyopathy (DCM) were previously analyzed, and the long noncoding RNA CHKB (choline kinase beta) divergent transcript (CHKB-DT) levels were found to be mostly downregulated in the heart. In this study, the function of CHKB-DT in DCM was determined. METHODS: Long noncoding RNA expression levels in the human heart tissues were measured via quantitative reverse transcription-polymerase chain reaction and in situ hybridization assays. A CHKB-DT heterozygous or homozygous knockout mouse model was generated using the clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9 system, and the adeno-associated virus with a cardiac-specific promoter was used to deliver the RNA in vivo. Sarcomere shortening was performed to assess the primary cardiomyocyte contractility. The Seahorse XF cell mitochondrial stress test was performed to determine the energy metabolism and ATP production. Furthermore, the underlying mechanisms were explored using quantitative proteomics, ribosome profiling, RNA antisense purification assays, mass spectrometry, RNA pull-down, luciferase assay, RNA-fluorescence in situ hybridization, and Western blotting. RESULTS: CHKB-DT levels were remarkably decreased in patients with DCM and mice with transverse aortic constriction-induced heart failure. Heterozygous knockout of CHKB-DT in cardiomyocytes caused cardiac dilation and dysfunction and reduced the contractility of primary cardiomyocytes. Moreover, CHKB-DT heterozygous knockout impaired mitochondrial function and decreased ATP production as well as cardiac energy metabolism. Mechanistically, ALDH2 (aldehyde dehydrogenase 2) was a direct target of CHKB-DT. CHKB-DT physically interacted with the mRNA of ALDH2 and fused in sarcoma (FUS) through the GGUG motif. CHKB-DT knockdown aggravated ALDH2 mRNA degradation and 4-HNE (4-hydroxy-2-nonenal) production, whereas overexpression of CHKB-DT reversed these molecular changes. Furthermore, restoring ALDH2 expression in CHKB-DT+/- mice alleviated cardiac dilation and dysfunction. CONCLUSIONS: CHKB-DT is significantly downregulated in DCM. CHKB-DT acts as an energy metabolism-associated long noncoding RNA and represents a promising therapeutic target against DCM.

Downregulation of Wtap causes dilated cardiomyopathy and heart failure.

RNA binding proteins have been shown to regulate heart development and cardiac diseases. However, the detailed molecular mechanisms is not known. In this study, we identified Wilms' tumor 1-associating protein (WTAP, a key regulatory protein of the m6A RNA methyltransferase complex) as a key regulator of heart function and cardiac diseases. WTAP is associated with heart development, and its expression is downregulated in both human and mice with heart failure. Cardiomyocyte-specific knockout of Wtap (Wtap-CKO) induces dilated cardiomyopathy, heart failure and neonatal death. Although WTAP deficiency in the heart decreases METTL3 (methyltransferase-like 3) protein levels, cardiomyocyte-specific overexpression of Mettl3 in Wtap-CKO mice does not rescue the phenotypes of Wtap-CKO mice. Instead, WTAP deficiency in the heart decreases chromatin accessibility in the promoter regions of Mef2a (myocyte enhancer factor-2α) and Mef2c, leading to reduced mRNA and protein levels of these genes and lower expression of their target genes. Conversely, WTAP directly binds to the promoter of the Mef2c gene and increases its promoter luciferase activity and expression. These data demonstrate that WTAP plays a key role in heart development and cardiac function by maintaining the chromatin accessibility of cardiomyocyte specific genes.

Biallelic PKP2 loss of function variants are associated with a lethal perinatal-onset biventricular dilated cardiomyopathy with excessive trabeculations and ventricular septal defects.

Homozygous plakophilin-2 (PKP2) variants have been identified as a cause of a lethal form of dilated cardiomyopathy with excessive trabeculations (DCM-ET) in three cases. We report three more cases from two families with homozygous pathogenic PKP2 variants and perinatal-onset, lethal DCM-ET. Identification of the genetic abnormalities played a key role in decision-making and family counselling in these cases. This case series supports the published evidence that biallelic loss of function PKP2 variants cause a lethal, perinatal-onset cardiomyopathy.

Skeletal Muscle Involvement in Patients With Truncations of Titin and Familial Dilated Cardiomyopathy.

BACKGROUND: Genetic variants in titin (TTN) are associated with dilated cardiomyopathy (DCM) and skeletal myopathy. However, the skeletal muscle phenotype in individuals carrying heterozygous truncating TTN variants (TTNtv), the leading cause of DCM, is understudied. OBJECTIVES: This study aimed to assess the skeletal muscle phenotype associated with TTNtv. METHODS: Participants with TTNtv were included in a cross-sectional study. Skeletal muscle fat fraction was evaluated by magnetic resonance imaging (compared with healthy controls and controls with non-TTNtv DCM). Muscle strength was evaluated by dynamometry and muscle biopsy specimens were analyzed. RESULTS: Twenty-five TTNtv participants (11 women, mean age 51 ± 15 years, left ventricular ejection fraction 45% ± 10%) were included (19 had DCM). Compared to healthy controls (n = 25), fat fraction was higher in calf (12.5% vs 9.9%, P = 0.013), thigh (12.2% vs 9.3%, P = 0.004), and paraspinal muscles (18.8% vs 13.9%, P = 0.008) of TTNtv participants. Linear mixed effects modelling found higher fat fractions in TTNtv participants compared to healthy controls (2.5%; 95% CI: 1.4-3.7; P < 0.001) and controls with non-TTNtv genetic DCM (n = 7) (1.5%; 95% CI: 0.2-2.8; P = 0.025). Muscle strength was within 1 SD of normal values. Biopsy specimens from 21 participants found myopathic features in 13 (62%), including central nuclei. Electron microscopy showed well-ordered Z-lines and T-tubuli but uneven and discontinuous M-lines and excessive glycogen depositions flanked by autophagosomes, lysosomes, and abnormal mitochondria with mitophagy. CONCLUSIONS: Mild skeletal muscle involvement was prevalent in patients with TTNtv. The phenotype was characterized by an increased muscle fat fraction and excessive accumulation of glycogen, possibly due to reduced autophagic flux. These findings indicate an impact of TTNtv beyond the heart.

Striated preferentially expressed gene deficiency leads to mitochondrial dysfunction in developing cardiomyocytes.

A deficiency of striated preferentially expressed gene (Speg), a member of the myosin light chain kinase family, results in abnormal myofibril structure and function of immature cardiomyocytes (CMs), corresponding with a dilated cardiomyopathy, heart failure and perinatal death. Mitochondrial development plays a role in cardiomyocyte maturation. Therefore, this study investigated whether Speg deficiency ( - / - ) in CMs would result in mitochondrial abnormalities. Speg wild-type and Speg-/- C57BL/6 littermate mice were utilized for assessment of mitochondrial structure by transmission electron and confocal microscopies. Speg was expressed in the first and second heart fields at embryonic (E) day 7.5, prior to the expression of mitochondrial Na+/Ca2+/Li+ exchanger (NCLX) at E8.5. Decreases in NCLX expression (E11.5) and the mitochondrial-to-nuclear DNA ratio (E13.5) were observed in Speg-/- hearts. Imaging of E18.5 Speg-/- hearts revealed abnormal mitochondrial cristae, corresponding with decreased ATP production in cells fed glucose or palmitate, increased levels of mitochondrial superoxide and depolarization of mitochondrial membrane potential. Interestingly, phosphorylated (p) PGC-1α, a key mediator of mitochondrial development, was significantly reduced in Speg-/- hearts during screening for targeted genes. Besides Z-line expression, Speg partially co-localized with PGC-1α in the sarcomeric region and was found in the same complex by co-immunoprecipitation. Overexpression of a Speg internal serine/threonine kinase domain in Speg-/- CMs promoted translocation of pPGC-1α into the nucleus, and restored ATP production that was abolished by siRNA-mediated silencing of PGC-1α. Our results demonstrate a critical role of Speg in mitochondrial development and energy metabolism in CMs, mediated in part by phosphorylation of PGC-1α.

Mutations in DNAJC19 cause altered mitochondrial structure and increased mitochondrial respiration in human iPSC-derived cardiomyocytes.

BACKGROUND: Dilated cardiomyopathy with ataxia (DCMA) is an autosomal recessive disorder arising from truncating mutations in DNAJC19, which encodes an inner mitochondrial membrane protein. Clinical features include an early onset, often life-threatening, cardiomyopathy associated with other metabolic features. Here, we aim to understand the metabolic and pathophysiological mechanisms of mutant DNAJC19 for the development of cardiomyopathy. METHODS: We generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) of two affected siblings with DCMA and a gene-edited truncation variant (tv) of DNAJC19 which all lack the conserved DnaJ interaction domain. The mutant iPSC-CMs and their respective control cells were subjected to various analyses, including assessments of morphology, metabolic function, and physiological consequences such as Ca2+ kinetics, contractility, and arrhythmic potential. Validation of respiration analysis was done in a gene-edited HeLa cell line (DNAJC19tvHeLa). RESULTS: Structural analyses revealed mitochondrial fragmentation and abnormal cristae formation associated with an overall reduced mitochondrial protein expression in mutant iPSC-CMs. Morphological alterations were associated with higher oxygen consumption rates (OCRs) in all three mutant iPSC-CMs, indicating higher electron transport chain activity to meet cellular ATP demands. Additionally, increased extracellular acidification rates suggested an increase in overall metabolic flux, while radioactive tracer uptake studies revealed decreased fatty acid uptake and utilization of glucose. Mutant iPSC-CMs also showed increased reactive oxygen species (ROS) and an elevated mitochondrial membrane potential. Increased mitochondrial respiration with pyruvate and malate as substrates was observed in mutant DNAJC19tv HeLa cells in addition to an upregulation of respiratory chain complexes, while cellular ATP-levels remain the same. Moreover, mitochondrial alterations were associated with increased beating frequencies, elevated diastolic Ca2+ concentrations, reduced sarcomere shortening and an increased beat-to-beat rate variability in mutant cell lines in response to ß-adrenergic stimulation. CONCLUSIONS: Loss of the DnaJ domain disturbs cardiac mitochondrial structure with abnormal cristae formation and leads to mitochondrial dysfunction, suggesting that DNAJC19 plays an essential role in mitochondrial morphogenesis and biogenesis. Moreover, increased mitochondrial respiration, altered substrate utilization, increased ROS production and abnormal Ca2+ kinetics provide insights into the pathogenesis of DCMA-related cardiomyopathy.

Challenges and Applications of Genetic Testing in Dilated Cardiomyopathy: Genotype, Phenotype and Clinical Implications. / Desafios e Aplicações dos Testes Genéticos na Cardiomiopatia Dilatada: Genótipo, Fenótipo e Implicações Clínicas.

Genetic tests for dilated cardiomyopathy (DCM) have a diagnostic yield of up to 40%, but there is significant genetic heterogeneity and other challenges, such as variable expressivity and incomplete penetrance. Pedigree analysis is essential for distinguishing between sporadic and familial DCM cases by assessing family history. Familial DCM yields higher results in genetic testing, but sporadic DCM does not rule out the possibility of a genetic cause. Some genes have specific phenotypes, with the Lamin gene ( LMNA ) being associated with a phenotype of malignant arrhythmias and advanced heart failure (HF). The presence of a causal genetic variant can also aid in prognostic evaluation, identifying more severe cases with lower rates of reverse remodeling (RR) compared to individuals with a negative genotype. Current guidelines recommend genetic evaluation and counseling for individuals with DCM, along with cascade screening in first-degree relatives in cases where one or more variants are identified, offering an opportunity for early diagnosis and treatment. Relatives with a positive genotype and negative phenotype are candidates for serial evaluation, with frequency varying by age. Genotype also assists in individualized recommendations for implantable cardioverter-defibrillator (ICD) placement and advice regarding physical activity and family planning. Ongoing studies are progressively elucidating the details of genotype/phenotype relationships for a large number of variants, making molecular genetics increasingly integrated into clinical practice.

Os testes genéticos para cardiomiopatia dilatada (CMD) apresentam uma positividade de até 40%, mas há uma grande heterogeneidade genética e outros desafios decorrentes de expressividade variável e penetrância incompleta. O heredograma é fundamental para diferenciar os casos de CMD esporádica e familiar, por meio da avaliação do histórico familiar. A CMD familiar apresenta um rendimento maior nos testes genéticos, mas a CMD esporádica não exclui a possibilidade de causa genética. Alguns genes têm fenótipos específicos, sendo o gene da Lamina ( LMNA ) o mais fortemente associado a um fenótipo de arritmias malignas e quadros de insuficiência cardíaca (IC) avançada. A presença de uma variante genética causal também pode ajudar na avaliação prognóstica, identificando quadros mais graves e com menores taxas de remodelamento reverso em comparação com indivíduos com genótipo negativo. As diretrizes atuais recomendam a avaliação e aconselhamento genético em indivíduos com CMD, além do rastreamento em cascata nos familiares de primeiro grau nos casos em que há uma ou mais variantes identificadas, sendo uma oportunidade para o diagnóstico e tratamento precoces. Familiares com genótipo positivo e fenótipo negativo são candidatos à avaliação seriada, com periodicidade que varia conforme a idade. O genótipo também auxilia na indicação individualizada de cardiodesfibrilador implantável e em recomendações quanto à atividade física e planejamento familiar. Estudos em curso esclarecem progressivamente os detalhes das relações genótipo/fenótipo de um grande número de variantes e fazem com que a genética molecular esteja cada vez mais presente na prática clínica.

Multiscale biophysical models of cardiomyopathies reveal complexities challenging existing dogmas.

Mutations in sarcomeric proteins, including myosin, cause a variety of cardiomyopathies. A prominent hypothesis has been that myosin mutations causing hypercontractility of the motor lead to hypertrophic cardiomyopathy, while those causing hypocontractility lead to dilated cardiomyopathy; however, recent biophysical studies using multiscale computational and experimental models have revealed complexities not captured by this hypothesis. We summarize recent publications in Biophysical Journal challenging this dogma and highlighting the need for multiscale modeling of these complex diseases.

GLUT1 Mediates the Metabolic Reprogramming and Inflammation of CCR2$

BACKGROUND: Macrophages expressing CC chemokine receptor 2 (CCR2) possess characteristics and performance akin to M1 polarized macrophages, which promote inflammation. Advanced heart failure (HF) patients with higher abundance of CCR2+ macrophages are more likely to experience adverse remodeling. The precise mechanism of CCR2+ macrophages in how they affect the progression of dilated cardiomyopathy remains unknown. METHODS: Cardiac biopsy samples from dilated cardiomyopathy patients (DCM) were used for immunohistochemistry and immunofluorescence staining. PCR is employed to identify the IL-1ß, IL-6, TNF-α, TGF-ß, MMP2, MMP9, PKM1, PKM1, GLUT1, GLUT2, GLUT3, GLUT4, PDK1, PFKFB3, PFK1 and HK2 mRNA expression of CCR2+ monocytes/macrophages from the peripheral blood of DCM patients. Seahorse was used to evaluate the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of CCR2+ monocytes/macrophages. 2-DG was used to simulate a lack of glucose. Lentivirus containing GLUT1 inhibitory sequence was used to knockdown GLUT1 gene expression of CCR2+ monocytes/macrophages. Western Blot and immunofluorescence staining was used to evaluate the expression of NLRP3. RESULTS: Immunostaining results of cardiac biopsy tissue from dilated cardiomyopathy (DCM) patients demonstrated that the progression to HF was associated with an increase in the number of CCR2+ macrophages. PCR results demonstrated that CCR2 monocytes and macrophages derived from the blood of DCM patients expressed elevated levels of inflammatory factors and up regulation of glycolysis related genes. In addition, OCR and glucose uptake experiments confirmed that increased glucose uptake of these cells was associated with greater inflammation and correlated with a worsening of cardiac function. limiting the glucose supply to CCR2+ monocytes and macrophages, or suppressing the activity of glucose transporter 1 (GLUT1) could reduce inflammation levels. CONCLUSIONS: These results suggest that CCR2+ monocytes and macrophages rely on metabolic reprogramming to trigger inflammatory response and contribute to myocardial injury and the progression of DCM.

MicroRNAs as novel biomarkers and potential therapeutic options for inflammatory cardiomyopathy.

AIMS: Inflammation of the heart is a complex biological and pathophysiological response of the immune system to a variety of injuries leading to tissue damage and heart failure. MicroRNAs (miRNAs) emerge as pivotal players in the development of numerous diseases, suggesting their potential utility as biomarkers for inflammation and as viable candidates for therapeutic interventions. The primary aim of this investigation was to pinpoint and assess particular miRNAs in individuals afflicted by virus-negative inflammatory dilated cardiomyopathy (DCMi). METHODS AND RESULTS: The study involved the analysis of 152 serum samples sourced from patients diagnosed with unexplained heart failure through endomyocardial biopsy. Among these samples, 38 belonged to DCMi patients, 24 to DCM patients, 44 to patients displaying inflammation alongside diverse viral infections, and 46 to patients solely affected by viral infections without concurrent inflammation. Additionally, serum samples from 10 healthy donors were included. The expression levels of 754 distinct miRNAs were evaluated using TaqMan OpenArray. MiR-1, miR-23, miR-142-5p, miR-155, miR-193, and miR-195 exhibited exclusive down-regulation solely in DCMi patients (P < 0.005). These miRNAs enabled effective differentiation between individuals with inflammation unlinked to viruses (DCMi) and all other participant groups (P < 0.005), boasting a specificity surpassing 86%. CONCLUSIONS: The identification of specific miRNAs offers a novel diagnostic perspective for recognizing intramyocardial inflammation within virus-negative DCMi patients. Furthermore, these miRNAs hold promise as potential candidates for tailored therapeutic strategies in the context of virus-negative DCMi.

A Kaposi's sarcoma-associated herpes virus-encoded microRNA contributes to dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is the leading cause of heart transplantation. By microRNA (miRNA) array, a Kaposi's sarcoma-associated herpes virus (KSHV)-encoded miRNA, kshv-miR-K12-1-5p, was detected in patients with DCM. The KSHV DNA load and kshv-miR-K12-1-5p level in plasma from 696 patients with DCM were measured and these patients were followed-up. Increased KSHV seropositivity and quantitative titers were found in the patients with DCM compared with the non-DCM group (22.0% versus 9.1%, p < 0.05; 168 versus 14 copies/mL plasma, p < 0.05). The risk of the individual end point of death from cardiovascular causes or heart transplantation was increased among DCM patients with the KSHV DNA seropositivity during follow-up (adjusted hazard ratio 1.38, 95% confidence interval 1.01-1.90; p < 0.05). In heart tissues, the KSHV DNA load was also increased in the heart from patients with DCM in comparison with healthy donors (1016 versus 29 copies/105 cells, p < 0.05). The KSHV and kshv-miR-K12-1-5p in DCM hearts were detected using immunofluorescence and fluorescence staining in situ hybridization. KSHV itself was exclusively detectable in CD31-positive endothelium, while kshv-miR-K12-1-5p could be detected in both endothelium and cardiomyocytes. Moreover, kshv-miR-K12-1-5p released by KSHV-infected cardiac endothelium could disrupt the type I interferon signaling pathway in cardiomyocytes. Two models of kshv-miR-K12-1-5p overexpression (agomiR and recombinant adeno-associated virus) were used to explore the roles of KSHV-encoded miRNA in vivo. The kshv-miR-K12-1-5p aggravated known cardiotropic viruses-induced cardiac dysfunction and inflammatory infiltration. In conclusion, KSHV infection was a risk factor for DCM, providing developmental insights of DCM involving virus and its miRNA ( https://clinicaltrials.gov . Unique identifier: NCT03461107).

An Alternative Mechanism of Subcellular Iron Uptake Deficiency in Cardiomyocytes.

BACKGROUND: Systemic defects in intestinal iron absorption, circulation, and retention cause iron deficiency in 50% of patients with heart failure. Defective subcellular iron uptake mechanisms that are independent of systemic absorption are incompletely understood. The main intracellular route for iron uptake in cardiomyocytes is clathrin-mediated endocytosis. METHODS: We investigated subcellular iron uptake mechanisms in patient-derived and CRISPR/Cas-edited induced pluripotent stem cell-derived cardiomyocytes as well as patient-derived heart tissue. We used an integrated platform of DIA-MA (mass spectrometry data-independent acquisition)-based proteomics and signaling pathway interrogation. We employed a genetic induced pluripotent stem cell model of 2 inherited mutations (TnT [troponin T]-R141W and TPM1 [tropomyosin 1]-L185F) that lead to dilated cardiomyopathy (DCM), a frequent cause of heart failure, to study the underlying molecular dysfunctions of DCM mutations. RESULTS: We identified a druggable molecular pathomechanism of impaired subcellular iron deficiency that is independent of systemic iron metabolism. Clathrin-mediated endocytosis defects as well as impaired endosome distribution and cargo transfer were identified as a basis for subcellular iron deficiency in DCM-induced pluripotent stem cell-derived cardiomyocytes. The clathrin-mediated endocytosis defects were also confirmed in the hearts of patients with DCM with end-stage heart failure. Correction of the TPM1-L185F mutation in DCM patient-derived induced pluripotent stem cells, treatment with a peptide, Rho activator II, or iron supplementation rescued the molecular disease pathway and recovered contractility. Phenocopying the effects of the TPM1-L185F mutation into WT induced pluripotent stem cell-derived cardiomyocytes could be ameliorated by iron supplementation. CONCLUSIONS: Our findings suggest that impaired endocytosis and cargo transport resulting in subcellular iron deficiency could be a relevant pathomechanism for patients with DCM carrying inherited mutations. Insight into this molecular mechanism may contribute to the development of treatment strategies and risk management in heart failure.

Whole-Exome Sequencing Identifies Homozygote Nonsense Variants in LMOD2 Gene Causing Infantile Dilated Cardiomyopathy.

As an essential component of the sarcomere, actin thin filament stems from the Z-disk extend toward the middle of the sarcomere and overlaps with myosin thick filaments. Elongation of the cardiac thin filament is essential for normal sarcomere maturation and heart function. This process is regulated by the actin-binding proteins Leiomodins (LMODs), among which LMOD2 has recently been identified as a key regulator of thin filament elongation to reach a mature length. Few reports have implicated homozygous loss of function variants of LMOD2 in neonatal dilated cardiomyopathy (DCM) associated with thin filament shortening. We present the fifth case of DCM due to biallelic variants in the LMOD2 gene and the second case with the c.1193G>A (p.W398*) nonsense variant identified by whole-exome sequencing. The proband is a 4-month male infant of Hispanic descent with advanced heart failure. Consistent with previous reports, a myocardial biopsy exhibited remarkably short thin filaments. However, compared to other cases of identical or similar biallelic variants, the patient presented here has an unusually late onset of cardiomyopathy during infancy. Herein, we present the phenotypic and histological features of this variant, confirm the pathogenic impact on protein expression and sarcomere structure, and discuss the current knowledge of LMOD2-related cardiomyopathy.

CCND1 Overexpression in Idiopathic Dilated Cardiomyopathy: A Promising Biomarker?

Cardiomyopathy, a disorder of electrical or heart muscle function, represents a type of cardiac muscle failure and culminates in severe heart conditions. The prevalence of dilated cardiomyopathy (DCM) is higher than that of other types (hypertrophic cardiomyopathy and restrictive cardiomyopathy) and causes many deaths. Idiopathic dilated cardiomyopathy (IDCM) is a type of DCM with an unknown underlying cause. This study aims to analyze the gene network of IDCM patients to identify disease biomarkers. Data were first extracted from the Gene Expression Omnibus (GEO) dataset and normalized based on the RMA algorithm (Bioconductor package), and differentially expressed genes were identified. The gene network was mapped on the STRING website, and the data were transferred to Cytoscape software to determine the top 100 genes. In the following, several genes, including VEGFA, IGF1, APP, STAT1, CCND1, MYH10, and MYH11, were selected for clinical studies. Peripheral blood samples were taken from 14 identified IDCM patients and 14 controls. The RT-PCR results revealed no significant differences in the expression of the genes APP, MYH10, and MYH11 between the two groups. By contrast, the STAT1, IGF1, CCND1, and VEGFA genes were overexpressed in patients more than in controls. The highest expression was found for VEGFA, followed by CCND1 (p < 0.001). Overexpression of these genes may contribute to disease progression in patients with IDCM. However, more patients and genes need to be analyzed in order to achieve more robust results.