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Titin is a giant sarcomeric protein that is involved in a large number of functions, with a primary role in skeletal and cardiac sarcomere organization and stiffness. The titin gene (TTN) is subject to various alternative splicing events, but in the region that is present at the M-line, the only exon that can be spliced out is Mex5, which encodes for the insertion sequence 7 (is7). Interestingly, in the heart, the majority of titin isoforms are Mex5+, suggesting a cardiac role for is7. Here, we performed comprehensive functional, histological, transcriptomic, microscopic and molecular analyses of a mouse model lacking the Ttn Mex5 exon (ΔMex5), and revealed that the absence of the is7 is causative for dilated cardiomyopathy. ΔMex5 mice showed altered cardiac function accompanied by increased fibrosis and ultrastructural alterations. Abnormal expression of excitation–contraction coupling proteins was also observed. The results reported here confirm the importance of the C-terminal region of titin in cardiac function and are the first to suggest a possible relationship between the is7 and excitation–contraction coupling. Finally, these findings give important insights for the identification of new targets in the treatment of titinopathies.

This study aims to provide new insights into transcriptome and miRome modifications occurring in cardiac reverse remodelling (RR) upon left ventricle pressure-overload relief in mice. Pressure-overload was established in seven-week-old C57BL/6J-mice by ascending aortic constriction. A debanding (DEB) surgery was performed seven weeks later in half of the banding group (BA). Two weeks later, cardiac function was evaluated through hemodynamics and echocardiography, and the hearts were collected for histology and small/bulk-RNA-sequencing. Pressure-overload relief was confirmed by the normalization of left-ventricle-end-systolic-pressure. DEB animals were separated into two subgroups according to the extent of cardiac remodelling at seven weeks and RR: DEB1 showed an incomplete RR phenotype confirmed by diastolic dysfunction persistence (E/e&rsquo, &ge, 16 ms) and increased myocardial fibrosis. At the same time, DEB2 exhibited normal diastolic function and fibrosis, presenting a phenotype closer to myocardial recovery. Nevertheless, both subgroups showed the persistence of cardiomyocytes hypertrophy. Notably, the DEB1 subgroup presented a more severe diastolic dysfunction at the moment of debanding than the DEB2, suggesting a different degree of cardiac remodelling. Transcriptomic and miRomic data, as well as their integrated analysis, revealed significant downregulation in metabolic and hypertrophic related pathways in DEB1 when compared to DEB2 group, including fatty acid &beta, oxidation, mitochondria L-carnitine shuttle, and nuclear factor of activated T-cells pathways. Moreover, extracellular matrix remodelling, glycan metabolism and inflammation-related pathways were up-regulated in DEB1. The presence of a more severe diastolic dysfunction at the moment of pressure overload-relief on top of cardiac hypertrophy was associated with an incomplete RR. Our transcriptomic approach suggests that a cardiac inflammation, fibrosis, and metabolic-related gene expression dysregulation underlies diastolic dysfunction persistence after pressure-overload relief, despite left ventricular mass regression, as echocardiographically confirmed.

Cardiovascular diseases are the number one cause of morbidity and mortality worldwide, but the underlying molecular mechanisms remain not well understood. Cardiomyopathies are primary diseases of the heart muscle and contribute to high rates of heart failure and sudden cardiac deaths. Here, we distinguished four different genetic cardiomyopathies based on gene expression signatures. In this study, RNA-Sequencing was used to identify gene expression signatures in myocardial tissue of cardiomyopathy patients in comparison to non-failing human hearts. Therefore, expression differences between patients with specific affected genes, namely \(\it LMNA\) (lamin A/C), \(\it RBM20\) (RNA binding motif protein 20), \(\it TTN\) (titin) and \(\it PKP2\) (plakophilin 2) were investigated. We identified genotype-specific differences in regulated pathways, Gene Ontology (GO) terms as well as gene groups like secreted or regulatory proteins and potential candidate drug targets revealing specific molecular pathomechanisms for the four subtypes of genetic cardiomyopathies. Some regulated pathways are common between patients with mutations in \(\it RBM20\) and \(\it TTN\) as the splice factor RBM20 targets amongst other genes \(\it TTN\), leading to a similar response on pathway level, even though many differentially expressed genes (DEGs) still differ between both sample types. The myocardium of patients with mutations in \(\it LMNA\) is widely associated with upregulated genes/pathways involved in immune response, whereas mutations in \(\it PKP2\) lead to a downregulation of genes of the extracellular matrix. Our results contribute to further understanding of the underlying molecular pathomechanisms aiming for novel and better treatment of genetic cardiomyopathies.

Background Heart failure is a major cause of morbidity and mortality worldwide, but the underlying molecular mechanisms remain not well defined. Reactive oxygen species (ROS) in heart failure (HF) alter multitudes of mitochondrial enzymes and metabolites, such as α-ketoglutarate and its oxidised form L-2-hydroxyglutarate (L-2HG). These metabolites are cofactors for ten eleven translocation (TET) enzymes that convert DNA's 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Aim We hypothesize that oxidative stress during heart failure alters mitochondrial function through epigenetic remodeling, and that reduction of oxidative stress will improve cardiac function. Methods and results Targeted LC-MS/MS analysis of dilated cardiomyopathy (DCM) hearts obtained from MLP−/− and WT littermate controls showed a significant increase in the oxidized metabolite L-2HG (∼30%, p=0.004), with significant reduction of multiple TCA cycle intermediates. RNA sequencing revealed a significant reduction in mRNA levels of IDH2 in human TTN related DCM and MLP−/− HF mice. The altered activity of IDH2 contributes to ROS production in these hearts and to the production of L-2HG. No alteration in the mitochondria structures was observed. HF biopsies show decreased TET activity most likely due to increased L-2HG levels. Whole genome single base pair 5mC and 5hmC deep sequencing of gDNA from explanted human DCM heart biopsies and MLP−/− mouse model revealed significantly altered global distribution of both 5mC and 5hmC in comparison to control samples. Genes involved in hypertrophy, such as Myh7 and Fhl2 were among the top genes with differential 5mC levels. Global loss of 5hmC level was observed, especially in the intronic regions of genes involved in redox hemostasis. Reducing oxidative stress in vivo in MLP−/− using a small molecule (AZ14117925) improves heart function (EF) by 13% [EF=(Treated:47,44%, Placebo: 34,54%), n=5 (males per group), p=0.0373, unpaired t-test]. Additional bioinformatic analysis revealed that reduction of ROS most likely leads to activation of TET and activation of pro-survival pathways, anti-oxidative stress response, and significantly less activation of apoptotic pathways. Conclusion Alterations in TCA cycle metabolites may underlie changes in DNA methylation and gene expression in end-stage human DCM and mouse models and indicate a role for epigenetic regulation of mitochondrial function in HF. NRF2 activation may pose a novel therapeutic approach to treat this devastating disease. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): AstraZeneca

International audience; Limb-girdle muscular dystrophy type 2A (LGMD2A or LGMDR1) is a neuromuscular disorder caused by mutations in the calpain 3 gene ( CAPN3 ). Previous experiments using adeno-associated viral (AAV) vector–mediated calpain 3 gene transfer in mice indicated cardiac toxicity associated with the ectopic expression of the calpain 3 transgene. Here, we performed a preliminary dose study in a severe double-knockout mouse model deficient in calpain 3 and dysferlin. We evaluated safety and biodistribution of AAV9-desmin-hCAPN3 vector administration to nonhuman primates (NHPs) with a dose of 3 × 10 13 viral genomes/kg. Vector administration did not lead to observable adverse effects or to detectable toxicity in NHP. Of note, the transgene expression did not produce any abnormal changes in cardiac morphology or function of injected animals while reaching therapeutic expression in skeletal muscle. Additional investigation on the underlying causes of cardiac toxicity observed after gene transfer in mice and the role of titin in this phenomenon suggest species-specific titin splicing. Mice have a reduced capacity for buffering calpain 3 activity compared to NHPs and humans. Our studies highlight a complex interplay between calpain 3 and titin binding sites and demonstrate an effective and safe profile for systemic calpain 3 vector delivery in NHP, providing critical support for the clinical potential of calpain 3 gene therapy in humans.

Background DNA methylation and hydroxymethylation plays critical role in important biological processes, including differentiation of tissues in the embryo and cellular response to different diseases and diverse environmental factors. The epigenetic landscape in heart failure might be altered. Purpose Our objective was to determine how Mybpc3 deficiency, which produces hypertrophic cardiomyopathy, affects epigenetic landscape, gene expression, and regulation. Methods We generated and analysed genome-wide DNA methylomes and hydroxymethylomes from cardiac tissues of 12-week old Mybpc3−/− mice and littermate controls, and performed whole genome RNA sequencing (RNA-seq) for gene expression and validated the findings using qPCR. Results Single base resolution revealed overall lower 5-mC level in Mybpc3 deficient mice. In deficient mice, different genic regions including transcription start site, exons, and introns, had low levels of 5-mC. Although there was no overall difference in 5-hmC content, knockout mice had lower levels of 5-hmC in the distal part of the genes (last exon, transcription termination site, and 3'-flanking regions). The 5-hmC enrichment in the intronic regions was associated with higher gene expression, whereas, the presence of 5-mC in the 5'-flanking regions was associated with lower gene expression in both knockout and wildtype mice. Ingenuity pathway analysis (IPA) of differentially expressed genes revealed overrepresentation of genes involved in axonal-guidance pathway. Tet activity was downregulated in Mybpc3−/− mice, and it may explain the overall difference of 5-mC in deficient mice. We also observed that Mybpc3 ablation affected alternative splicing of Myh6 and Myh7. Conclusion This study establishes that knocking out of Mybpc3 changes epigenetic landscape in cardiac tissue, which is tightly linked to gene expression and regulation. Acknowledgement/Funding LeDucq 13CVD04, Hjärt och Lungfonden (Sweden)

Background Mice lacking muscle LIM protein (Mlp/Cspr3 −/−) develop dilated cardiomyopathy (DCM). Previous work established this model to be amenable to improvements in cardiac function by genetic ablation of phospholamban (PLN). Purpose To test the hypothesis that therapeutic reductions of PLN would similarly improve cardiac function, Mlp KO mice were administered an antisense oligonucleotide (ASO) targeting PLN. Methods Echocardiography measurements of ejection fraction (EF), end-diastolic volume (EDV) and end-systolic volume (ESV) were performed before and after treatment. In addition, global transcriptome profiling using 3'RNA-seq was performed to identify gene expression changes in diseased Mlp KO mice and following PLN ASO treatments. Mlp KO mice with ejection fraction (EF%) of less than 45% (median, 37.6%; interquartile range, 32.2–42.0%) were treated with vehicle (n=10) or PLN ASO (n=9) for 4 weeks. Results Three subcutaneous injections of PLN ASO were administered to Mlp KO mice resulting in 50–70% PLN reductions. Echocardiography performed at study end revealed improvements of EF (60±8 vs. 46±12%), ESV (31±11 vs. 56±21μl) and EDV (79±22 vs. 100±25μl) with PLN ASO treatment. Corrected for baseline values, PLN ASO treatment improved all echocardiographic measurements (p In conclusion, antisense inhibition of PLN reduced functional and transcriptional indices of heart failure in a DCM model. In view of the failed CUPID trials, a gene therapy approach to improve SERCA2a activity, targeting PLN with ASO may be advantageous due to a likely more robust pharmacological profile.

AIMS: A 25-base pair deletion in the cardiac myosin binding protein-C (cMyBP-C) gene (MYBPC3), proposed to skip exon 33, modifies the C10 domain (cMyBP-C(ΔC10mut)) and is associated with hypertrophic cardiomyopathy (HCM) and heart failure, affecting approximately 100 million South Asians. However, the molecular mechanisms underlying the pathogenicity of cMyBP-C(ΔC10mut)in vivo are unknown. We hypothesized that expression of cMyBP-C(ΔC10mut) exerts a poison polypeptide effect leading to improper assembly of cardiac sarcomeres and the development of HCM. METHODS AND RESULTS: To determine whether expression of cMyBP-C(ΔC10mut) is sufficient to cause HCM and contractile dysfunction in vivo, we generated transgenic (TG) mice having cardiac-specific protein expression of cMyBP-C(ΔC10mut) at approximately half the level of endogenous cMyBP-C. At 12 weeks of age, significant hypertrophy was observed in TG mice expressing cMyBP-C(ΔC10mut) (heart weight/body weight ratio: 4.43 ± 0.11 mg/g non-transgenic (NTG) vs. 5.34 ± 0.25 mg/g cMyBP-C(ΔC10mut), P

In this study, we investigated the role of DNA methylation [5-methylcytosine (5mC)] and 5-hydroxymethylcytosine (5hmC), epigenetic modifications that regulate gene activity, in dilated cardiomyopathy (DCM). A MYBPC3 mutant mouse model of DCM was compared with wild type and used to profile genomic 5mC and 5hmC changes by Chip-seq, and gene expression levels were analyzed by RNA-seq. Both 5mC-altered genes (957) and 5hmC-altered genes (2,022) were identified in DCM hearts. Diverse gene ontology and KEGG pathways were enriched for DCM phenotypes, such as inflammation, tissue fibrosis, cell death, cardiac remodeling, cardiomyocyte growth, and differentiation, as well as sarcomere structure. Hierarchical clustering of mapped genes affected by 5mC and 5hmC clearly differentiated DCM from wild-type phenotype. Based on these data, we propose that genomewide 5mC and 5hmC contents may play a major role in DCM pathogenesis. NEW & NOTEWORTHY Our data demonstrate that development of dilated cardiomyopathy in mice is associated with significant epigenetic changes, specifically in intronic regions, which, when combined with gene expression profiling data, highlight key signaling pathways involved in pathological cardiac remodeling and heart contractile dysfunction.

Heart failure (HF) is a major cause of morbidity and mortality worldwide, highlighting an urgent need for novel treatment options, despite recent improvements. Aberrant Ca2+ handling is a key feature of HF pathophysiology. Restoring the Ca2+ regulating machinery is an attractive therapeutic strategy supported by genetic and pharmacological proof of concept studies. Here, we study antisense oligonucleotides (ASOs) as a therapeutic modality, interfering with the PLN/SERCA2a interaction by targeting Pln mRNA for downregulation in the heart of murine HF models. Mice harboring the PLN R14del pathogenic variant recapitulate the human dilated cardiomyopathy (DCM) phenotype; subcutaneous administration of PLN-ASO prevents PLN protein aggregation, cardiac dysfunction, and leads to a 3-fold increase in survival rate. In another genetic DCM mouse model, unrelated to PLN (Cspr3/Mlp−/−), PLN-ASO also reverses the HF phenotype. Finally, in rats with myocardial infarction, PLN-ASO treatment prevents progression of left ventricular dilatation and improves left ventricular contractility. Thus, our data establish that antisense inhibition of PLN is an effective strategy in preclinical models of genetic cardiomyopathy as well as ischemia driven HF., Heart failure is a major cause of morbidity and mortality worldwide. Here the authors show that subcutaneous administration of antisense oligonucleotides targeting PLN is an effective strategy in preclinical models of genetic cardiomyopathy and ischemia-driven heart failure.