by University of Maryland School of Medicine

cygb2 mutant phenotype presents organ laterality defects. a CRISPR/Cas9 mediated genome editing of cygb2. Two different gRNA were targeted to exon 1 (denoted by red text) and resulted in 4 bp and 1 bp frame shift mutations (beginning in the blue shaded region of the predicted protein structure) named cygb2^801a and cygb2^801b, respectively. The eight globin protein helices (labeled A-H) are represented by boxes, with out-of-frame amino acids shaded blue. b Whole mount 3D confocal projections (right) of wt and cygb2^pt801a tg(fli1eGFP^y1) hearts at 4 days post fertilization (dpf) with schematic (left) representing the heart morphology and direction of blood flow. V – ventricle, A – atrium, Y – yolk. Scale bar = 20 µm. c, d Quantification of the percentage of embryos with a left-sided heart loop in cygb2^801a and cygb2^801b. Means are ± SD (n = 6–7, each n representing an independent experiment consisting of 50 embryos). Student’s t test, two-tailed, **P < 0.01, ***P < 0.001. e Representative in situ hybridization images of the cygb2^801a laterality phenotype. southpaw (spaw), 16 somites, dorsal view; lefty2 (lft2), 22 h post fertilization (hpf), dorsal view; myosin light chain 7 (myl7), 96 hpf, ventral view; and foxa3, 2 dpf, dorsal view. Green arrow heads indicate the liver and blue arrow heads point to the pancreas. Scale bars = 100 μm. f Quantification of the percentage of embryos with right, straight/bilateral or left sided expression of spaw, lft2, mly7 or foxa3. The total number of embryos analyzed is shown in red above the graph. The Chi-squared test was used to determine statistical significance. Source data are provided as source data file. Credit: Nature Communications (2023). DOI: 10.1038/s41467-023-43544-0

In a study led by the University of Maryland School of Medicine, researchers discovered for the first time that a certain kind of protein similar to hemoglobin, called cytoglobin, plays an important role in the development of the heart. Specifically, it affects the correct left-right pattern of the heart and other asymmetric organs.

The findings, published today in the journal Nature Communications, could eventually lead to the development of new therapeutic interventions to alter the processes that lead to these defects.

The team used CRISPR gene editing technologies to knock out the cytoglobin gene in zebrafish. The lack of cytoglobin caused the development of embryos with a mirrored heart, meaning the heart had a reversed left-right pattern. In humans, cytoglobin is involved in nitric oxide processes, a compound that helps regulate healthy blood flow to organs.

Study co-senior author Mark T. Gladwin, MD, the John Z. and Akiko K. Bowers Distinguished Professor and Dean, University of Maryland School of Medicine, and Vice President for Medical Affairs, University of Maryland, Baltimore, has been researching the effects of nitric oxide on blood vessels for more than 20 years including in this recent study finding.

"Since its discovery two decades ago, cytoglobin has been found to be expressed in nearly all human tissues, but the mechanisms of how this protein functions were largely unknown," said Dr. Gladwin. "We know that cytoglobin can play a role in modulating and maintaining nitric oxide levels, but our new finding indicates that it positively regulates NO production to ensure proper cilia function, and its absence can lead to major laterality abnormalities of organs."

Zebrafish with mutant gene lacking cytoglobin. Heart defects are seen with heart on the right side instead of the left side with blood flow looping to the left instead of the right. Credit: University of Maryland School of Medicine

To conduct the study, the research team knocked out the gene for cytoglobin in zebrafish and was amazed to see that it led to dramatic defects in the structure and location of organs in developing embryos. The heart, for example, was located on the right side of the fish instead of the left, with a looping to the left instead of the right.

"We found that cytoglobin plays a vital role in the structure and function of tiny hair-like structures called cilia, which determine the asymmetry and proper development of organs," said study senior author Paola Corti, Ph.D., Assistant Professor of Biochemistry and Molecular Biology at UMSOM.

This is the first time cytoglobin—or any of the globin proteins like hemoglobin—has been found to be involved in fetal development and that a paucity could be linked to birth defects. It's also the first time that cytoglobin has been linked to cilia function. Such a finding could open the door for the development of therapeutics for rare birth defects that affect the movement of cilia.

About 1 in every 10,000 to 30,000 people are born with Primary Ciliary Dykinesia (PCD), a rare disease that affects the cilia and can cause breathing issues from thickened mucus clogging airways. "Kartagener's syndrome is a form of PCD and is known to cause the type of heart defects seen in the zebrafish where the heart is abnormally positioned to the right and rotated," said Dr. Corti. "There is no cure for this condition, just surgery to fix any heart defects and treatments to manage symptoms."

While certain genes have been identified that are known to cause about 70 percent of PCD cases, cytoglobin could play a key role in the 30 percent of cases with no known genetic cause.

"We found the phenotype and connected the dots to cilia. In the presence of cytoglobin, we could track the function of the protein and how it led to proper cilia function and organ development. In the absence, we saw these defects," said Elizabeth Rochon, Ph.D., first author of the study and Assistant Professor of Medicine at UMSOM.

More information: Elizabeth R. Rochon et al, Cytoglobin regulates NO-dependent cilia motility and organ laterality during development, Nature Communications (2023). DOI: 10.1038/s41467-023-43544-0

Journal information: Nature Communications 

Provided by University of Maryland School of Medicine