Sabharwal, Ankit

Functional analyses of mitochondria have been hampered by few effective approaches to manipulate mitochondrial DNA (mtDNA) and a lack of existing animal models. Recently a TALE-derived base editor was shown to induce C-to-T (or G-to-A) sequence changes in mtDNA. We report here the FusX TALE Base Editor (FusXTBE) to facilitate broad-based access to TALE mitochondrial base editing technology. TALE Writer is a de novo in silico design tool to map potential mtDNA base editing sites. FusXTBE was demonstrated to function with comparable activity to the initial base editor in human cells in vitro . Zebrafish embryos were used as a pioneering in vivo test system, with FusXTBE inducing 90+% editing efficiency in mtDNA loci as an example of near-complete induction of mtDNA heteroplasmy in vivo . Gene editing specificity as precise as a single nucleotide was observed for a protein-coding gene. Nondestructive genotyping enables single-animal mtDNA analyses for downstream biological functional genomic applications. FusXTBE is a new gene editing toolkit for exploring important questions in mitochondrial biology and genetics.

Myocardial infarction (MI) remains a leading cause of mortality worldwide, yet effective cardioprotective strategies remain limited in clinical settings. Vascular endothelial growth factor B (VEGFB) has emerged as a promising therapeutic candidate in MI, but the role of its co-receptor, Neuropilin-1 (NRP1), in cardiomyocyte (CM) survival under ischemic stress remains poorly understood. Here, we investigated VEGFB-NRP1 signaling using an in vivo zebrafish model of cardiac injury as well as in vitro hypoxia models in CMs. We demonstrated that VEGFB overexpression conferred protection against ischemic injury and enhanced cardiac regeneration in the zebrafish heart. Mechanistically, we showed that VEGFB treatment enhances CM viability through reducing reactive oxygen species (ROS), ferroptosis activation, and preserving mitochondrial integrity. We also demonstrated that NRP1 knockdown in the CMs abolished the VEGFB-mediated protective effects, indicating the significant role of NRP1 signaling in VEGFB-induced cardioprotective effects in MI. Lastly, using transcriptome analysis, we confirmed that VEGFB induces anti-apoptotic and anti-ferroptosis gene programs in CMs in response to hypoxic stress. Collectively, our findings provide mechanistic insight into cell death activation pathways, including ferroptosis, in response to ischemic stress and further validate the therapeutic potential of VEGFB in promoting CM survival in ischemic heart disease.

The rapid advancement of nuclear and mitochondrial genomic editing tools has created an urgent need for efficient, nonlethal larval genotyping methods in zebrafish ( Danio rerio ) research. This study optimizes and validates a nondestructive proteinase K digestion method for mitochondrial and nuclear DNA genotyping while characterizing its impact on larval survival and gene expression. Using optimized protocol parameters, we demonstrate successful amplification of different mitochondrial and nuclear genetic loci with consistently high sensitivity. Molecular validation through PCR, restriction fragment length polymorphism analysis, and Sanger sequencing confirmed the specificity and reliability of the extracted DNA. The method successfully detected C-to-T base edits in the mt-tl1 gene introduced using the FusX TALE Base editor system, demonstrating its applicability to gene editing studies. Both 48-well and optimized 96-well formats were used, enabling this approach to be deployed at scale. This optimized method enables researchers to correlate genotypes with phenotypes in longitudinal studies while maintaining specimen viability, particularly valuable for investigating early-onset mitochondrial diseases, and utilizes standard laboratory equipment and reagents, facilitating widespread adoption in zebrafish research while adhering to ethical principles in reducing animal mortality.

Abstract Background Myocardial infarction (MI) remains a leading cause of mortality worldwide. Recent studies suggest a cardioprotective role for vascular endothelial growth factor-B (VEGF-B) in MI. However, the molecular mechanisms of VEGF-B-mediated signaling via its co-receptor Neuropilin-1 (NRP1) in MI are poorly understood. In this study, we investigated the intricate signaling mechanisms involving VEGF-B and NRP1 in cardiomyocytes (CMs) using ischemic injury as a model of MI. And secondly, we further validated the protective role of VEFG-B in ischemic heart disease, shedding light on their roles not only in cardiac function but also in its therapeutic potential. Methods In this study, we utilized both in vitro and in vivo approaches to elucidate the role of VEGF-B and NRP1 signaling in MI and how it manifests a protective role in mitochondrial functions and cardiac regeneration following ischemic injury. We used two different cardiomyocyte cell lines, H9c2 (rat ventricular cardiomyocytes) and HL-1 (mouse ventricular cardiomyocytes), and induced hypoxia conditions, using either 1% oxygen or 200µM cobalt chloride (CoCl 2 ) to mimic the myocardial infarction-induced ischemic injury in the heart. In addition, we developed a novel heat shock inducible zebrafish model of a cardiomyocyte-specific VEGF-B overexpression system to further examine the protective role of VEGF-B in vivo . Results Our findings indicate that both VEGF-B and NRP1 are predominantly expressed in heart tissue compared to other tissues, and their expression is altered in response to hypoxia/ischemic injury. Our results demonstrate that VEGF-B treatment prior to hypoxia enhances cardiomyocyte survival, while NRP1 knockdown abolishes this protective effect, highlighting a prominent role of NRP1 signaling in VEGF-B-mediated cardio protection. Furthermore, we found that VEGF-B promotes cardiomyocyte survival by improving mitochondrial function, as evidenced by reduced oxidative stress and ROS accumulation, decreased oxidative stress, preserved mitochondrial membrane potential, and increased ATP levels. Lastly, using our VEGF-B transgenic zebrafish model, we demonstrated that VEGF-B overexpression in zebrafish cardiomyocytes protects the heart from ischemic injury and enhances cardiac regeneration in an NRP1-dependent manner. Conclusion Our study has uncovered an important role of VEGF-B-NRP1 signaling axis in VEGF-B mediated cell survival and in beneficial mitochondrial functions in the CMs. Importantly, we also demonstrated that VEGF-B is protective against ischemic injury in vivo using a novel zebrafish model Novelty and Significance What Is Known? Myocardial infarction (MI) results in mitochondrial dysfunction, cardiomyocyte death, and adverse ventricular remodeling. Vascular Endothelial Growth Factor B (VEGF-B) is traditionally associated with cardiac metabolism, survival, vascular biology, particularly in angiogenesis and lipid metabolism. The neurophilin-1 (NRP1) receptor, a co-receptor for VEGF-B, is expressed in cardiomyocytes and implicated in mitochondrial signaling pathways. Emerging evidence suggests that VEGF-B may influence mitochondrial integrity and function under stress conditions. What New Information Does This Article Contribute? VEGF-B exerts a protective effect on cardiomyocyte mitocondria following ischemic injury by preserving mitochondrial function and reducing oxidative stress. NRP1 receptor as a key mediator of VEGF-B modulates mitochondrial homeostasis in cardiomyocytes. VEGF-B signaling can attenuate apoptosis and enhance mitochondrial bioenergetics post-MI, suggesting a novel cardioprotective mechanism. Clinical Implication: These findings suggest that therapeutic strategies aimed at enhancing VEGF-B/NRP1 signaling may improve mitochondrial function and cardiac recovery after MI, offering a novel avenue for limiting heart failure progression in ischemic heart disease.

Mitochondria are critical organelles that form networks within our cells, generate energy dynamically, contribute to diverse cell and organ function, and produce a variety of critical signaling molecules, such as cortisol. This intracellular microbiome can differ between cells, tissues, and organs. Mitochondria can change with disease, age, and in response to the environment. Single nucleotide variants in the circular genomes of human mitochondrial DNA are associated with many different life-threatening diseases. Mitochondrial DNA base editing tools have established novel disease models and represent a new possibility toward personalized gene therapies for the treatment of mtDNA-based disorders.

The clinical and largely unpredictable heterogeneity of phenotypes in patients with mitochondrial disorders demonstrates the ongoing challenges in the understanding of this semi-autonomous organelle in biology and disease. Previously, we used the gene-breaking transposon to create 1200 transgenic zebrafish strains tagging protein-coding genes (Ichino et al., 2020), including the lrpprc locus. Here, we present and characterize a new genetic revertible animal model that recapitulates components of Leigh Syndrome French Canadian Type (LSFC), a mitochondrial disorder that includes diagnostic liver dysfunction. LSFC is caused by allelic variations in the LRPPRC gene, involved in mitochondrial mRNA polyadenylation and translation. lrpprc zebrafish homozygous mutants displayed biochemical and mitochondrial phenotypes similar to clinical manifestations observed in patients, including dysfunction in lipid homeostasis. We were able to rescue these phenotypes in the disease model using a liver-specific genetic model therapy, functionally demonstrating a previously under-recognized critical role for the liver in the pathophysiology of this disease.

Mitochondria are a dynamic eukaryotic innovation that play diverse roles in biology and disease. The mitochondrial genome is remarkably conserved in all vertebrates, encoding the same 37-gene set and overall genomic structure, ranging from 16,596 base pairs (bp) in the teleost zebrafish (Danio rerio) to 16,569 bp in humans. Mitochondrial disorders are amongst the most prevalent inherited diseases, affecting roughly 1 in every 5000 individuals. Currently, few effective treatments exist for those with mitochondrial ailments, representing a major unmet patient need. Mitochondrial dysfunction is also a common component of a wide variety of other human illnesses, ranging from neurodegenerative disorders such as Huntington’s disease and Parkinson’s disease to autoimmune illnesses such as multiple sclerosis and rheumatoid arthritis. The electron transport chain (ETC) component of mitochondria is critical for mitochondrial biology and defects can lead to many mitochondrial disease symptoms. Here, we present a publicly available collection of genetic mutants created in highly conserved, nuclear-encoded mitochondrial genes in Danio rerio. The zebrafish system represents a potentially powerful new opportunity for the study of mitochondrial biology and disease due to the large number of orthologous genes shared with humans and the many advanced features of this model system, from genetics to imaging. This collection includes 15 mutant lines in 13 different genes created through locus-specific gene editing to induce frameshift or splice acceptor mutations, leading to predicted protein truncation during translation. Additionally, included are 11 lines created by the random insertion of the gene-breaking transposon (GBT) protein trap cassette. All these targeted mutant alleles truncate conserved domains of genes critical to the proper function of the ETC or genes that have been implicated in human mitochondrial disease. This collection is designed to accelerate the use of zebrafish to study many different aspects of mitochondrial function to widen our understanding of their role in biology and human disease.

One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression shows previously undocumented expression of 35% and 90% of cloned genes at 2 and 4 days post-fertilization, respectively. Further, investigated alleles regularly show 99% gene-specific mRNA knockdown. Homozygous GBT animals in ryr1b , fras1 , tnnt2a , edar and hmcn1 phenocopied established mutants. 204 cloned lines trapped diverse proteins, including 64 orthologs of human disease-associated genes with 40 as potential new disease models. Severely reduced skeletal muscle Ca 2+ transients in GBT ryr1b homozygous animals validated the ability to explore molecular mechanisms of genetic diseases. This GBT system facilitates novel functional genome annotation towards understanding cellular and molecular underpinnings of vertebrate biology and human disease.