Redox and epigenetic modulators regulate cardiac function and remodeling in health and disease

Oxidative species are a divergent group of cellular metabolites with a wide variety of functions. Together with reductants, they regulate almost all cellular functions, from mediating cellular communications to catalyzing a variety of biochemical reactions, and further to posttranslationally modifying proteins. The past decades’ focus on oxidative species as injurious byproducts associated with diseases have not yielded any clinical success. For example, attempts to improve heart function by antioxidative treatments have rather, in some cases, had adverse effects on heart failure. Therefore, there is unmet need for a change in the way we perceive redox biology, namely, to replace the traditional view on oxidants as unambiguous foes with more openminded perspective on the broad functions of the redox system and the novel mechanisms that regulate the endogenous antioxidative capacity. An urge for unbiased approaches is further supported by the recent technical advances in multi omics, which have enabled the exploration of complex mechanisms beyond traditional boundaries. In our recent manuscript on BioRxiv (Elbeck et al., 2022), on which this thesis is largely based, we present evidence using multipronged omics that mitochondrial isocitrate dehydrogenase 2 (IDH2) governs an extensive redox-regulatory mechanism in cardiac mitochondria. We found that IDH2 together with nuclear factor erythroid 2-related factor 2 (NRF2) coordinates a novel antioxidative mechanism through a feedforward cycle involving 2-oxoglutarate (2OG) and L2hydroxyglutarate (L2HG). We further found that this redox cycle regulates gene expression through an unconventional mechanism involving intronic DNA hydroxymethylation. We explored the possible implications of these findings for the treatment of heart failure, taking into consideration the previously failed clinical trials. We obtained evidence for sexual dimorphism in mice in which females showed a more robust antioxidative defense reflecting on their heart failure phenotype: a less severe dilated cardiomyopathy (DCM) compared to males. We tested our hypothesis using a novel pharmacological compound AZ925, which activated the NRF2 pathway. Our conclusion is that enhancing the antioxidative capacity has a positive impact on cardiac function only when endogenous antioxidative capacity is limited, highlighting new possibilities for precision medicine. In the literature review part of this thesis, I aimed to explore literature beyond the protective role of the redox system. Here, I dig deeply into the multifaceted essential—but overlooked— functions of this system. I also aimed to explain my reasoning behind the design and interpretation of some of the data presented in Elbeck et al., 2022. Moreover, I further explored if data from cases of patients with DCM were potentially supportive of my hypothesis (Project I). In Project II, I have investigated the importance of miR-208b-3p, which is a highly induced micro-RNA (miR) in the myocardia of patients with DCM. I propose that miR208b-3p plays a role in the cardiac reverse remodeling observed in some patients with heart failure as a potential redoxmiR, which represents one of the arms of the redox system discussed in this monograph. Project III does not deal directly with redox biology, but it is rather related to the concept of translatable genetic information beyond the canonical protein coding and translational reading frames via alternative splicing. We propose the existence of multiple isoforms of muscle lim protein (MLP) translated at extremely low levels from same Mlp pre-mRNA as the full length MPL protein, even in Mlp-/- animals that have a deletion in Mlp exon2. These isoforms retain some of the functional domains of their full-length protein, and therefore may mediate distinct functions. The overall goal of my work has been to use recent technical advances to explore biological mechanisms beyond some of its preconceived boundaries, and thereby to unveil novel molecular mechanisms that could ultimately lead to improved personalized and precise treatments of several diseases, including redox therapies for heart failure

Phospholamban antisense oligonucleotides improve cardiac function in murine cardiomyopathy

Heart failure (HF) is a major cause of morbidity and mortality worldwide, highlighting an urgent need for novel treatment options, despite recent improvements. Aberrant Ca(2+) handling is a key feature of HF pathophysiology. Restoring the Ca(2+) 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

Titin M-line insertion sequence 7 is required for proper cardiac function in mice

International audienceABSTRACT 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

The Degree of Cardiac Remodelling before Overload Relief Triggers Different Transcriptome and miRome Signatures during Reverse Remodelling (RR)—Molecular Signature Differ with the Extent of RR

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’ ≥ 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 β-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

Distinct Myocardial Transcriptomic Profiles of Cardiomyopathies Stratified by the Mutant Genes

Sielemann K, Elbeck Z, Gärtner A, et al. Distinct Myocardial Transcriptomic Profiles of Cardiomyopathies Stratified by the Mutant Genes. Genes. 2020;11(12): 1430.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 LMNA (lamin A/C), RBM20 (RNA binding motif protein 20), TTN (titin) and 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 RBM20 and TTN as the splice factor RBM20 targets amongst other genes 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 LMNA is widely associated with upregulated genes/pathways involved in immune response, whereas mutations in 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

Mosaic deletion of claudin-5 reveals rapid non-cell-autonomous consequences of blood-brain barrier leakage

Summary: Claudin-5 (CLDN5) is an endothelial tight junction protein essential for blood-brain barrier (BBB) formation. Abnormal CLDN5 expression is common in brain disease, and knockdown of Cldn5 at the BBB has been proposed to facilitate drug delivery to the brain. To study the consequences of CLDN5 loss in the mature brain, we induced mosaic endothelial-specific Cldn5 gene ablation in adult mice (Cldn5iECKO). These mice displayed increased BBB permeability to tracers up to 10 kDa in size from 6 days post induction (dpi) and ensuing lethality from 10 dpi. Single-cell RNA sequencing at 11 dpi revealed profound transcriptomic differences in brain endothelial cells regardless of their Cldn5 status in mosaic mice, suggesting major non-cell-autonomous responses. Reactive microglia and astrocytes suggested rapid cellular responses to BBB leakage. Our study demonstrates a critical role for CLDN5 in the adult BBB and provides molecular insight into the consequences and risks associated with CLDN5 inhibition

Species-specific titin splicing regulates cardiotoxicity associated with calpain 3 gene therapy for limb-girdle muscular dystrophy 2A

International audienceLimb-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 × 1013 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

Distinct myocardial transcriptomic profiles of cardiomyopathies stratified by the mutant genes

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

Epigenetic modulators link mitochondrial redox homeostasis to cardiac function in a sex-dependent manner.

Acknowledgements: We acknowledge all people who have contributed to this study, either by providing funds or technical assistance. We want to particularly acknowledge Mattias Svensson, Department of Medicine Huddinge, for his valuable intellectual input and feedback, Oscar Franzén for his valuable input in the epigenetic bioinformatic analysis, Cristobal Dos Remedios and Amy Li for the provision of the human heart samples from the Sydney biobank and for his scientific input to the manuscript. Byambajav Buyandelger for her valuable input and for helping in preparing samples for TEM. Anne-Laure Lainé, for preparing AZ925, David Brodin at the BEA facility, KI, Huddinge, for his support in analyzing the ShRNA-KD RNA seq data, Kjell Hultenby for performing the TEM and quantifying the images obtained, Sara Fernandez Leon and Charlotte Webster for their technical assistance, and Joseph W DePierre for scientific English editing of the manuscript. We would like to acknowledge the Single-cell core facility at the Flemingsberg campus (SICOF) at Karolinska Institutet (KI) for their sequencing. This facility is supported by grants from the KI Department of Medicine (MedH) and KI Infrastructure, as well as the infrastructure for the Strategic Research Area (SFO) on Stem Cells and Regenerative Medicine. Fluorescence microscopy was performed at the Live Cell Imaging Core facility/Nikon Center of Excellence at Karolinska Institutet, with support from the Swedish Research Council, KI infrastructure, and the Centre for Innovative Medicine. We also acknowledge Gisele Miranda for her input in the analysis of fluorescence images. The BioImage Informatics Facility for carrying out image analysis is funded by SciLifeLab, the National Microscopy Infrastructure, NMI (VR-RFI 2019-00217), and the Chan-Zuckerberg Initiative. The Proteomics Biomedicum core facility of Karolinska Institutet for performing the mass spectrometric analysis. Antibodies against VDAC, COX4I1, and NDUFB8 were a kind gift from Peter Rehling, Göttingen, and we thank Berkan Arslan for performing the Western blotting. We also acknowledge Hanna Ebrel from the Institute of Pharmacology and Toxicology, University of Würzburg, Germany, for her invaluable assistance in preparing and differentiating hiPSC-CMs. C.M. is supported by the German Research Foundation (DFG; SFB 894, TRR-219; Ma 2528/7-1) and the German Ministry of Education and Research (BMBF, 01EO1504). N.O. is supported financially by the Knut and Alice Wallenberg Foundation as part of the National Bioinformatics Infrastructure Sweden at SciLifeLab.While excessive production of reactive oxygen species (ROS) is a characteristic hallmark of numerous diseases, clinical approaches that ameliorate oxidative stress have been unsuccessful. Here, utilizing multi-omics, we demonstrate that in cardiomyocytes, mitochondrial isocitrate dehydrogenase (IDH2) constitutes a major antioxidative defense mechanism. Paradoxically reduced expression of IDH2 associated with ventricular eccentric hypertrophy is counterbalanced by an increase in the enzyme activity. We unveil redox-dependent sex dimorphism, and extensive mutual regulation of the antioxidative activities of IDH2 and NRF2 by a feedforward network that involves 2-oxoglutarate and L-2-hydroxyglutarate and mediated in part through unconventional hydroxy-methylation of cytosine residues present in introns. Consequently, conditional targeting of ROS in a murine model of heart failure improves cardiac function in sex- and phenotype-dependent manners. Together, these insights may explain why previous attempts to treat heart failure with antioxidants have been unsuccessful and open new approaches to personalizing and, thereby, improving such treatment