Single-Cell RNA Sequencing Reveals Cardiac Fibroblast-Specific Transcriptomic Changes in Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM) is the most common cause of heart failure, with a complex aetiology involving multiple cell types. We aimed to detect cell-specific transcriptomic alterations in DCM through analysis that leveraged recent advancements in single-cell analytical tools. Single-cell RNA sequencing (scRNA-seq) data from human DCM cardiac tissue were subjected to an updated bioinformatic workflow in which unsupervised clustering was paired with reference label transfer to more comprehensively annotate the dataset. Differential gene expression was detected primarily in the cardiac fibroblast population. Bulk RNA sequencing was performed on an independent cohort of human cardiac tissue and compared with scRNA-seq gene alterations to generate a stratified list of higher-confidence, fibroblast-specific expression candidates for further validation. Concordant gene dysregulation was confirmed in TGFß-induced fibroblasts. Functional assessment of gene candidates showed that AEBP1 may play a significant role in fibroblast activation. This unbiased approach enabled improved resolution of cardiac cell-type-specific transcriptomic alterations in DCM.

Unraveling the Etiology of Dilated Cardiomyopathy through Differential miRNA-mRNA Interactome.

Dilated cardiomyopathy (DCM) encompasses various acquired or genetic diseases sharing a common phenotype. The understanding of pathogenetic mechanisms and the determination of the functional effects of each etiology may allow for tailoring different therapeutic strategies. MicroRNAs (miRNAs) have emerged as key regulators in cardiovascular diseases, including DCM. However, their specific roles in different DCM etiologies remain elusive. Here, we applied mRNA-seq and miRNA-seq to identify the gene and miRNA signature from myocardial biopsies from four patients with DCM caused by volume overload (VCM) and four with ischemic DCM (ICM). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were used for differentially expressed genes (DEGs). The miRNA-mRNA interactions were identified by Pearson correlation analysis and miRNA target-prediction programs. mRNA-seq and miRNA-seq were validated by qRT-PCR and miRNA-mRNA interactions were validated by luciferase assays. We found 112 mRNAs and five miRNAs dysregulated in VCM vs. ICM. DEGs were positively enriched for pathways related to the extracellular matrix (ECM), mitochondrial respiration, cardiac muscle contraction, and fatty acid metabolism in VCM vs. ICM and negatively enriched for immune-response-related pathways, JAK-STAT, and NF-kappa B signaling. We identified four pairs of negatively correlated miRNA-mRNA: miR-218-5p-DDX6, miR-218-5p-TTC39C, miR-218-5p-SEMA4A, and miR-494-3p-SGMS2. Our study revealed novel miRNA-mRNA interaction networks and signaling pathways for VCM and ICM, providing novel insights into the development of these DCM etiologies.

Viral myocarditis in combination with genetic cardiomyopathy as a cause of sudden death. An autopsy series.

Sudden cardiac death (SCD) is a major public health issue worldwide. In the young (< 40 years of age), genetic cardiomyopathies and viral myocarditis, sometimes in combination, are the most frequent, but underestimated, causes of SCD. Molecular autopsy is essential for prevention. Several studies have shown an association between genetic cardiomyopathies and viral myocarditis, which is probably underestimated due to insufficient post-mortem investigations. We report on four autopsy cases illustrating the pathogenesis of these combined pathologies. In two cases, a genetic hypertrophic cardiomyopathy was diagnosed in combination with Herpes Virus Type 6 (HHV6) and/or Parvovirus-B19 (PVB19) in the heart. In the third case, autopsy revealed a dilated cardiomyopathy and virological analyses revealed acute myocarditis caused by three viruses: PVB19, HHV6 and Epstein-Barr virus. Genetic analyses revealed a mutation in the gene coding for desmin. The fourth case illustrated a channelopathy and a PVB19/HHV6 coinfection. Our four cases illustrate the highly probable deleterious role of cardiotropic viruses in the occurrence of SCD in subjects with genetic cardiomyopathies. We discuss the pathogenetic link between viral myocarditis and genetic cardiomyopathy. Molecular autopsy is essential in prevention of these SCD, and a close collaboration between cardiologists, pathologists, microbiologists and geneticians is mandatory.

Generation of a pluripotent stem cell line (UMGi270-A) and a corresponding CRISPR/Cas9 modified isogenic control (UMGi270-A-1) from a patient with sudden onset dilated cardiomyopathy harboring a FLNC p.R2187P mutation.

Filamin C (FLNC) is a highly important actin crosslinker and multi-adaptor protein in striated skeletal and cardiac muscle. Mutations have been linked to a range of cardiomyopathy types. Here, we generated induced pluripotent stem cells (iPSC) from a patient with dilated cardiomyopathy (DCM) harboring a new, unique heterozygous FLNC mutation p.R2187P. From this patient-specific iPSC line, a corresponding isogenic control line was created by CRISPR/Cas9 genome editing. Both, the patient-specific and isogenic-control iPSC maintained full pluripotency, genomic integrity, and in vitro differentiation capacity. All iPSC lines differentiate into iPSC-cardiomyocytes, hence providing the possibility to study the pathogenesis of FLNC-mediated DCM further.

Integrated machine learning and multimodal data fusion for patho-phenotypic feature recognition in iPSC models of dilated cardiomyopathy.

Integration of multiple data sources presents a challenge for accurate prediction of molecular patho-phenotypic features in automated analysis of data from human model systems. Here, we applied a machine learning-based data integration to distinguish patho-phenotypic features at the subcellular level for dilated cardiomyopathy (DCM). We employed a human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model of a DCM mutation in the sarcomere protein troponin T (TnT), TnT-R141W, compared to isogenic healthy (WT) control iPSC-CMs. We established a multimodal data fusion (MDF)-based analysis to integrate source datasets for Ca2+ transients, force measurements, and contractility recordings. Data were acquired for three additional layer types, single cells, cell monolayers, and 3D spheroid iPSC-CM models. For data analysis, numerical conversion as well as fusion of data from Ca2+ transients, force measurements, and contractility recordings, a non-negative blind deconvolution (NNBD)-based method was applied. Using an XGBoost algorithm, we found a high prediction accuracy for fused single cell, monolayer, and 3D spheroid iPSC-CM models (≥92 ± 0.08 %), as well as for fused Ca2+ transient, beating force, and contractility models (>96 ± 0.04 %). Integrating MDF and XGBoost provides a highly effective analysis tool for prediction of patho-phenotypic features in complex human disease models such as DCM iPSC-CMs.

Mst4, a novel cardiac STRIPAK complex-associated kinase, regulates cardiomyocyte growth and survival and is upregulated in human cardiomyopathy.

Myocardial failure is associated with adverse remodeling, including loss of cardiomyocytes, hypertrophy, and alterations in cell-cell contacts. Striatin-interacting phosphatase and kinase (STRIPAK) complexes and their mammalian STE20-like kinase 4 (Mst4) have been linked to development of different diseases. The role and targets of Mst4 in cardiomyocytes have not been investigated yet. Multitissue immunoblot experiments show highly enriched Mst4 expression in rodent hearts. Analyses of human biopsy samples from patients suffering from dilated cardiomyopathy revealed that Mst4 is upregulated (5- to 8-fold p < 0.001) compared with nonfailing controls. Increased abundance of Mst4 could also be detected in mouse models of cardiomyopathy. We confirmed that Mst4 interacts with STRIPAK components in neonatal rat ventricular cardiomyocytes, indicating that STRIPAK is present in the heart. Immunofluorescence stainings and molecular interaction studies revealed that Mst4 is localized to the intercalated disc and interacts with several intercalated disc proteins. Overexpression of Mst4 in cardiomyocytes results in hypertrophy compared with controls. In adult rat cardiomyocytes, Mst4 overexpression increases cellular and sarcomeric fractional shortening (p < 0.05), indicating enhanced contractility. Overexpression of Mst4 also inhibits apoptosis shown by reduction of cleaved caspase3 (-69%, p < 0.0001), caspase7 (-80%, p < 0.0001), and cleaved Parp1 (-27%, p < 0.001). To elucidate potential Mst4 targets, we performed phosphoproteomics analyses in neonatal rat cardiomyocytes after Mst4 overexpression and inhibition. The results revealed target candidates of Mst4 at the intercalated disc. We identified Mst4 as a novel cardiac kinase that is upregulated in cardiomyopathy-regulating cardiomyocyte growth and survival.

Familial childhood onset, slowly progressive myopathy plus cardiomyopathy expands the phenotype related to variants in the TTN gene.

This report describes a novel TTN -related phenotype in two brothers, both affected by a childhood onset, very slowly progressive myopathy with cores, associated with dilated cardiomyopathy only in their late disease stages. Clinical exome sequencing documented in both siblings the heterozygous c.2089A>T and c.19426+2T>A variants in TTN. The c.2089A>T, classified in ClinVar as possibly pathogenic, introduces a premature stop codon in exon 14, whereas the c.19426+2T>A affects TTN alternative splicing. The unfeasibility of segregation studies prevented us from establishing the inheritance mode of the muscle disease in this family, although the lack of any reported muscle or heart symptoms in both parents might support an autosomal recessive transmission. In this view, the occurrence of cardiomyopathy in both probands might be related to the c.2089A>T truncating variant in exon 14, and the childhood onset, slowly progressive myopathy to the c.19426+2T>A splicing variant, possibly allowing translation of an almost full length TTN protein.

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.

Temporal progression of replacement and interstitial fibrosis in optimally managed dilated cardiomyopathy patients: A prospective study.

BACKGROUND: To prospectively examine the dynamic evolution of fibrotic processes within a one-year in patients with dilated cardiomyopathy (DCM). METHODS: Between May 2019 and September 2020, 102 DCM patients (mean age 45.2 ± 11.8 years, EF 29.9 ± 11.6%) underwent cardiac magnetic resonance (CMR-1). After 13.9 ± 2.9 months, 92 of these patients underwent a follow-up CMR (CMR-2). Replacement fibrosis was assessed via late gadolinium enhancement (LGE), quantified in terms of LGE mass and extent. Interstitial fibrosis was evaluated via T1-mapping and expressed as extracellular volume fraction (ECV). This data, along with left ventricular (LV) mass, facilitated the calculation of LV matrix and cellular volumes. RESULTS: At CMR-1, LGE was present in 45 patients (48.9%), whereas at CMR-2 LGE was detected in 46 (50%) (p = 0.88). Although LGE mass remained stable, LGE extent increased from 2.18 ± 4.1% to 2.7 ± 4.6% (p < 0.01). Conversely, ECV remained unchanged [27.7% (25.5-31.3) vs. 26.7% (24.5-29.9); p = 0.19]; however, LV matrix and cell volumes exhibited a noteworthy regression. During a subsequent follow-up of 19.2 ± 9 months (spanning from CMR-2 to April 30th, 2023), the composite primary outcome (all-cause mortality, HTX, LVAD or heart failure worsening) was evident in 18 patients. Only the LV matrix volume index at follow-up was an independent predictor of outcome (OR 1.094; 95%CI 1.004-1.192; p < 0.05). CONCLUSIONS: In optimally managed DCM patients, both replacement and interstitial fibrosis remained stable over the course of one year. In contrast, LV matrix and cell volumes displayed significant regression. LV matrix volume index at 12-month follow-up was found to be an independent predictor of outcome in DCM.

Deciphering the role of SMARCA4 in cardiac disorders: Insights from single-cell studies on dilated cardiomyopathy and coronary heart disease.

BACKGROUND: Dilated cardiomyopathy (DCM) and coronary heart disease (CHD) stand as two of the foremost causes of mortality. However, the comprehensive comprehension of the regulatory mechanisms governing DCM and CHD remains limited, particularly from the vantage point of single-cell transcriptional analysis. METHOD: We used the GSE121893 dataset from the GEO database, analyzing single-cell expressions with tools like DropletUtils, Seurat, and Monocle. We also utilized the GSVA package for comparing gene roles in DCM and CHD, Finally, we conducted qRT-PCR and Western blot analyses to measure the expression levels of SMARCA4, Col1A1, Col3A1 and α-SMA, and the role of SMARCA4 on fibroblasts were explored by EdU and Transwell assay. RESULTS: Our analysis identified six cell types in heart tissue, with fibroblasts showing the most interaction with other cells. DEGs in fibroblasts were linked to muscle development and morphogenesis. Pseudotime analysis revealed the dynamics of fibroblast changes in both the normal and disease groups and many transcription factors (TFs) potentially involved in this process. Among these TFs, SMARCA4 which was translated into protein BRG1, showed the most significantly difference. In vivo experiments have demonstrated that SMARCA4 indeed promoted fibroblasts proliferation and migration. CONCLUSION: This study provides a clearer understanding of cell-type dynamics in heart diseases, emphasizing the role of fibroblasts and the significance of SMARCA4 in their function. Our results offer insights into the cellular mechanisms underlying DCM and CHD, potentially guiding future therapeutic strategies.

Cardiac phenotype in adolescents and young adults with long-chain 3-hydroxyacyl CoA dehydrogenase (LCHAD) deficiency.

PURPOSE: Long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency (LCHADD) is a rare fatty acid oxidation disorder characterized by recurrent episodes of metabolic decompensation and rhabdomyolysis, as well as retinopathy, peripheral neuropathy, and cardiac involvement, such as infantile dilated cardiomyopathy. Because LCHADD patients are surviving longer, we sought to characterize LCHADD-associated major cardiac involvement in adolescence and young adulthood. METHODS: A retrospective cohort of 16 adolescent and young adult participants with LCHADD was reviewed for cardiac phenotype. RESULTS: Major cardiac involvement occurred in 9 of 16 participants, including sudden death, out-of-hospital cardiac arrest, acute cardiac decompensations with heart failure and/or in-hospital cardiac arrest, end-stage dilated cardiomyopathy, and moderate restrictive cardiomyopathy. Sudden cardiac arrest was more common in males and those with a history of infant cardiomyopathy. CONCLUSION: The cardiac manifestations of LCHADD in adolescence and early adulthood are complex and distinct from the phenotype seen in infancy. Life-threatening arrhythmia occurs at substantial rates in LCHADD, often in the absence of metabolic decompensation or rhabdomyolysis. The potential risk factors identified here-male sex and history of infant cardiomyopathy-may hint at strategies for risk stratification and possibly the prevention of these events.

Role of TBX20 Truncating Variants in Dilated Cardiomyopathy and Left Ventricular Noncompaction.

BACKGROUND: Less than 40% of patients with dilated cardiomyopathy (DCM) have a pathogenic/likely pathogenic genetic variant identified. TBX20 has been linked to congenital heart defects; although an association with left ventricular noncompaction (LVNC) and DCM has been proposed, it is still considered a gene with limited evidence for these phenotypes. This study sought to investigate the association between the TBX20 truncating variant (TBX20tv) and DCM/LVNC. METHODS: TBX20 was sequenced by next-generation sequencing in 7463 unrelated probands with a diagnosis of DCM or LVNC, 22 773 probands of an internal comparison group (hypertrophic cardiomyopathy, channelopathies, or aortic diseases), and 124 098 external controls (individuals from the gnomAD database). Enrichment of TBX20tv in DCM/LVNC was calculated, cosegregation was determined in selected families, and clinical characteristics and outcomes were analyzed in carriers. RESULTS: TBX20tv was enriched in DCM/LVNC (24/7463; 0.32%) compared with internal (1/22 773; 0.004%) and external comparison groups (4/124 098; 0.003%), with odds ratios of 73.23 (95% CI, 9.90-541.45; P<0.0001) and 99.76 (95% CI, 34.60-287.62; P<0.0001), respectively. TBX20tv was cosegregated with DCM/LVNC phenotype in 21 families for a combined logarythm of the odds score of 4.53 (strong linkage). Among 57 individuals with TBX20tv (49.1% men; mean age, 35.9±20.8 years), 41 (71.9%) exhibited DCM/LVNC, of whom 14 (34.1%) had also congenital heart defects. After a median follow-up of 6.9 (95% CI, 25-75:3.6-14.5) years, 9.7% of patients with DCM/LVNC had end-stage heart failure events and 4.8% experienced malignant ventricular arrhythmias. CONCLUSIONS: TBX20tv is associated with DCM/LVNC; congenital heart defect is also present in around one-third of cases. TBX20tv-associated DCM/LVNC is characterized by a nonaggressive phenotype, with a low incidence of major cardiovascular events. TBX20 should be considered a definitive gene for DCM and LVNC and routinely included in genetic testing panels for these phenotypes.

Allelic heterogeneity of TTNtv dilated cardiomyopathy can be modeled in adult zebrafish.

Allelic heterogeneity (AH) has been noted in truncational TTN-associated (TTNtv-associated) dilated cardiomyopathy (DCM); i.e., mutations affecting A-band-encoding exons are pathogenic, but those affecting Z-disc-encoding exons are likely benign. The lack of an in vivo animal model that recapitulates AH hinders the deciphering of the underlying mechanism. Here, we explored zebrafish as a candidate vertebrate model by phenotyping a collection of zebrafish ttntv alleles. We noted that cardiac function and sarcomere structure were more severely disrupted in ttntv-A than in ttntv-Z homozygous embryos. Consistently, cardiomyopathy-like phenotypes were present in ttntv-A but not ttntv-Z adult heterozygous mutants. The phenotypes observed in ttntv-A alleles were recapitulated in null mutants with the full titin-encoding sequences removed. Defective autophagic flux, largely due to impaired autophagosome-lysosome fusion, was also noted only in ttntv-A but not in ttntv-Z models. Moreover, we found that genetic manipulation of ulk1a restored autophagy flux and rescued cardiac dysfunction in ttntv-A animals. Together, our findings presented adult zebrafish as an in vivo animal model for studying AH in TTNtv DCM, demonstrated TTN loss of function is sufficient to trigger ttntv DCM in zebrafish, and uncovered ulk1a as a potential therapeutic target gene for TTNtv DCM.

CRISPR Activation Reverses Haploinsufficiency and Functional Deficits Caused by TTN Truncation Variants.

BACKGROUND: TTN truncation variants (TTNtvs) are the most common genetic lesion identified in individuals with dilated cardiomyopathy, a disease with high morbidity and mortality rates. TTNtvs reduce normal TTN (titin) protein levels, produce truncated proteins, and impair sarcomere content and function. Therapeutics targeting TTNtvs have been elusive because of the immense size of TTN, the rarity of specific TTNtvs, and incomplete knowledge of TTNtv pathogenicity. METHODS: We adapted CRISPR activation using dCas9-VPR to functionally interrogate TTNtv pathogenicity and develop a therapeutic in human cardiomyocytes and 3-dimensional cardiac microtissues engineered from induced pluripotent stem cell models harboring a dilated cardiomyopathy-associated TTNtv. We performed guide RNA screening with custom TTN reporter assays, agarose gel electrophoresis to quantify TTN protein levels and isoforms, and RNA sequencing to identify molecular consequences of TTN activation. Cardiomyocyte epigenetic assays were also used to nominate DNA regulatory elements to enable cardiomyocyte-specific TTN activation. RESULTS: CRISPR activation of TTN using single guide RNAs targeting either the TTN promoter or regulatory elements in spatial proximity to the TTN promoter through 3-dimensional chromatin interactions rescued TTN protein deficits disturbed by TTNtvs. Increasing TTN protein levels normalized sarcomere content and contractile function despite increasing truncated TTN protein. In addition to TTN transcripts, CRISPR activation also increased levels of myofibril assembly-related and sarcomere-related transcripts. CONCLUSIONS: TTN CRISPR activation rescued TTNtv-related functional deficits despite increasing truncated TTN levels, which provides evidence to support haploinsufficiency as a relevant genetic mechanism underlying heterozygous TTNtvs. CRISPR activation could be developed as a therapeutic to treat a large proportion of TTNtvs.

Immune cells and related cytokines in dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a non-ischemic cardiomyopathy involving one or more underlying etiologies. It is characterized by structural and functional dysfunction of the myocardium, potentially leading to fibrosis and ventricular remodeling, and an elevated risk of heart failure (HF). Although the pathogenesis of DCM remains unknown, compelling evidence suggests that DCM-triggered immune cells and inflammatory cascades play a crucial role in the occurrence and development of DCM. Various factors are linked to myocardial damage, inducing aberrant activation of the immune system and sustained inflammatory responses in DCM. The investigation of the immunopathogenesis of DCM also contributes to discovering new biomarkers and therapeutic targets. This review examines the roles of immune cells and related cytokines in DCM pathogenesis and explores immunotherapy strategies in DCM.

Identification of patients with nonischemic dilated cardiomyopathy at risk of malignant ventricular arrhythmias: insights from cardiac magnetic resonance feature tracking.

BACKGROUND: Patients with nonischemic dilated cardiomyopathy (NIDCM) are prone to arrhythmias, and the cause of mortality in these patients is either end-organ dysfunction due to pump failure or malignant arrhythmia-related death. However, the identification of patients with NIDCM at risk of malignant ventricular arrhythmias (VAs) is challenging in clinical practice. The aim of this study was to evaluate whether cardiovascular magnetic resonance feature tracking (CMR-FT) could help in the identification of patients with NIDCM at risk of malignant VAs. METHODS: A total of 263 NIDCM patients who underwent CMR, 24-hour Holter electrocardiography (ECG) and inpatient ECG were retrospectively evaluated. The patients with NIDCM were allocated to two subgroups: NIDCM with VAs and NIDCM without VAs. From CMR-FT, the global peak radial strain (GPRS), global longitudinal strain (GPLS), and global peak circumferential strain (GPCS) were calculated from the left ventricle (LV) model. We investigated the possible predictors of NIDCM combined with VAs by univariate and multivariate logistic regression analyses. RESULTS: The percent LGE (15.51 ± 3.30 vs. 9.62 ± 2.18, P < 0.001) was higher in NIDCM patients with VAs than in NIDCM patients without VAs. Furthermore, the NIDCM patients complicated with VAs had significantly lower GPCS than the NIDCM patients without VAs (- 5.38 (- 7.50, - 4.22) vs.-9.22 (- 10.73, - 8.19), P < 0.01). Subgroup analysis based on LGE negativity showed that NIDCM patients complicated with VAs had significantly lower GPRS, GPCS, and GPLS than NIDCM patients without VAs (P < 0.05 for all). Multivariate analysis showed that both GPCS and %LGE were independent predictors of NIDCM combined with VAs. CONCLUSIONS: CMR global strain can be used to identify NIDCM patients complicated with VAs early, specifically when LGE is not present. GPCS < - 13.19% and %LGE > 10.37% are independent predictors of NIDCM combined with VAs.

CD4+TEM cells drive the progression from acute myocarditis to dilated cardiomyopathy in CVB3-induced BALB/c mice.

Acute viral myocarditis can progress to chronic myocarditis leading to dilated cardiomyopathy (DCM). Persistent CD4+ T-cell-mediated autoimmunity triggered by infection plays a critical role in this progression. Increasing evidence demonstrates that effector memory CD4+T (CD4+TEM) cells, a subset of memory CD4+ T cells, are crucial pathogenic mediators of many autoimmune diseases. However, the role of CD4+TEM cells during the progression from acute viral myocarditis to DCM remains unknown. In this study, we observed an increase in CD4+TEM cells both in the periphery and the heart, and memory CD4+ T cells were the predominant sources of IL-17A and IFN-γ among inflamed heart-infiltrating CD4+ T cells during the progression from acute myocarditis to chronic myocarditis and DCM in CVB3-induced BALB/c mice. Moreover, splenic CD4+TEM cells sorted from DCM mice induced by CVB3 were found to respond to cardiac self-antigens ex vivo. Additionally, adoptive transfer experiments substantiated their pathogenic impact, inducing sustained myocardial inflammation, tissue fibrosis, cardiac injury, and impairment of cardiac systolic function in vivo. Our findings illustrate that long-lived CD4+TEM cells are important contributors to the progression from acute viral myocarditis into DCM.

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.

Sex Differences in the Clinical Presentation and Natural History of Dilated Cardiomyopathy.

BACKGROUND: Biological sex has a diverse impact on the cardiovascular system. Its influence on dilated cardiomyopathy (DCM) remains unresolved. OBJECTIVES: This study aims to investigate sex-specific differences in DCM presentation, natural history, and prognostic factors. METHODS: The authors conducted a prospective observational cohort study of DCM patients assessing baseline characteristics, cardiac magnetic resonance imaging, biomarkers, and genotype. The composite outcome was cardiovascular mortality or major heart failure (HF) events. RESULTS: Overall, 206 females and 398 males with DCM were followed for a median of 3.9 years. At baseline, female patients had higher left ventricular ejection fraction, smaller left ventricular volumes, less prevalent mid-wall myocardial fibrosis (23% vs 42%), and lower high-sensitivity cardiac troponin I than males (all P < 0.05) with no difference in time from diagnosis, age at enrollment, N-terminal pro-B-type natriuretic peptide levels, pathogenic DCM genetic variants, myocardial fibrosis extent, or medications used for HF. Despite a more favorable profile, the risk of the primary outcome at 2 years was higher in females than males (8.6% vs 4.4%, adjusted HR: 3.14; 95% CI: 1.55-6.35; P = 0.001). Between 2 and 5 years, the effect of sex as a prognostic modifier attenuated. Age, mid-wall myocardial fibrosis, left ventricular ejection fraction, left atrial volume, N-terminal pro-B-type natriuretic peptide, high-sensitivity cardiac troponin I, left bundle branch block, and NYHA functional class were not sex-specific prognostic factors. CONCLUSIONS: The authors identified a novel paradox in prognosis for females with DCM. Female DCM patients have a paradoxical early increase in major HF events despite less prevalent myocardial fibrosis and a milder phenotype at presentation. Future studies should interrogate the mechanistic basis for these sex differences.

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.