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.

Linked-read sequencing promises a one-method approach for genome-wide insights including single nucleotide variants (SNVs), structural variants, and haplotyping. We introduce Barcode Linked Reads (BLR), an open-source haplotyping pipeline capable of handling millions of barcodes and data from multiple linked-read technologies including DBS, 10× Genomics, TELL-seq and stLFR. Running BLR on DBS linked-reads yielded megabase-scale phasing with low (<0.2%) switch error rates. Of 13616 protein-coding genes phased in the GIAB benchmark set (v4.2.1), 98.6% matched the BLR phasing. In addition, large structural variants showed concordance with HPRC-HG002 reference assembly calls. Compared to diploid assembly with PacBio HiFi reads, BLR phasing was more continuous when considering switch errors. We further show that integrating long reads at low coverage (∼10×) can improve phasing contiguity and reduce switch errors in tandem repeats. When compared to Long Ranger on 10× Genomics data, BLR showed an increase in phase block N50 with low switch-error rates. For TELL-Seq and stLFR linked reads, BLR generated longer or similar phase block lengths and low switch error rates compared to results presented in the original publications. In conclusion, BLR provides a flexible workflow for comprehensive haplotype analysis of linked reads from multiple platforms.

Precision medicine is increasingly applied in the cancer clinic, adapting treatment to genomic alterations of the tumor. However, whether alterations disrupt the function of a protein can depend on if both alleles of a gene are altered. While massively parallel sequencing technologies can identify sequence aberrations, they are limited in resolving the corresponding haplotype information. In this proof-of-concept case study, we applied the linked-read droplet barcode sequencing (DBS) technology to resolve the haplotype complexity of colorectal cancer genomes on paired tumor and normal samples. Several cancer-related genes carried multiple mutations in either one or both haplotypes. Additionally, a number of haplotype-resolved large structural variants and copy number alterations were detected and phased with short somatic variants. Nearly all characterized oncogenic pathways harbored some of the identified short somatic variants. The study demonstrates that linked-read DBS technology can characterize complex genetic variations in a haplotype context and may provide essential information for personalized approaches.

Excessive production of reactive oxygen species (ROS) is characteristic of numerous diseases, but most studies in this area have not considered the impact of endogenous antioxidative defenses. Here, utilizing multi-omics, we demonstrate that in cardiomyocytes mitochondrial isocitrate dehydrogenase (IDH2) constitutes a major antioxidant defense. In both male and female mice and humans the paradoxical reduction in expression of IDH2 associated with heart failure is compensated for by an increase in the enzyme’s activity. We describe extensive mutual regulation of the antioxidant activities of IDH2 and NRF2 by a network involving 2-oxoglutarate and L2-hydroxyglutarate and mediated in part through unconventional hydroxymethylation of cytosine residues present in introns. Conditional targeting of ROS in a murine model of heart failure improves cardiac function. 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. Highlights Paradoxical downregulation of mitochondrial isocitrate dehydrogenase (IDH2) in response to oxidative stress leads to the discovery of a robust antioxidative defense in the heart. An antioxidative loop involving IDH2 coordinates other antioxidative defenses, such as NRF2. This loop produces epigenetic modifications that link oxidative stress to mitochondrial function. The conclusion that enhancing antioxidative capacity improves cardiac function only when the endogenous capacity is insufficient opens new approaches to individualized treatment of patients with heart failure.

Background: Cardiac stress induces MYH6 -to- MYH7 remodeling and activates redox-defense programs, but the regulatory mechanisms that distinguish adaptive from maladaptive stress responses remain incompletely defined. We investigated the role of miR-208b-3p, an intronic MYH7 microRNA, in cardiac stress adaptation. Methods and Results: We integrated myocardial transcriptomic and small RNA sequencing from stress-adapted Mlp ^−/− mice and human cardiomyopathy to identify stress-adaptive programs associated with preserved myocardial viability and complemented these analyses with in vivo miR-208b-3p inhibition and cellular gain-of-function experiments. Mlp ^−/− hearts exhibited marked Myh7 induction accompanied by an ≈20-fold increase in miR-208b-3p, whereas this coordinated response was blunted in human end-stage dilated and ischemic cardiomyopathies. In vivo inhibition of miR-208b-3p selectively reduced Myh7 , without altering Myh6 or baseline cardiac function. This reduction in Myh7 occurred without induction of established Myh7 transcriptional repressors, and miR-208b-3p gain-of-function similarly reduced MYH7 while suppressing several canonical repressors, indicating that its relationship with MYH7 extends beyond the established repressor pathway. miR-208b-3p inhibition activated oxidative stress, NRF2, autophagy, and cell-survival pathways. Oxidative stress transiently suppressed miR-208b-3p, with rebound induction after stress removal, whereas antioxidant treatment increased its expression. Pharmacological NRF2 activation did not alter miR-208b-3p, whereas miR-208b-3p gain-of-function reduced oxidative stress-associated apoptotic signaling and suppressed ATM, a predicted target and regulator of cellular stress responses, supporting an upstream role for miR-208b-3p in stress adaptation. Conclusions: MiR-208b-3p is a dynamic component of cardiac stress adaptation that links myosin remodeling with redox and survival responses. Its regulation of ATM and association with NRF2-related stress responses support an upstream role in coordinating cellular adaptation to cardiac stress. Graphical