How does a developing heart know how big to grow?
Northwestern Medicine scientists have uncovered an unexpected part of the answer: lymphatic vessels, long known for transporting fluid and immune cells, also send signals to different cardiac cell types that help control heart growth. Understanding this communication may ultimately have implications for heart repair after a heart attack and could inform future approaches for investigating congenital heart defects, the study authors said.
"Some of the mechanisms that help control how the heart grows during development may also be important when the adult heart is injured," said senior study author Guillermo Oliver, the Thomas D. Spies Professor of Lymphatic Metabolism at Northwestern University Feinberg School of Medicine. "Understanding that relationship could ultimately give us new insight into how the heart responds to and repairs itself after a heart attack."
The study was published earlier this month in the journal Genes and Development.
The scientists used single-cell RNA sequencing and genetically engineered mouse models to identify a signaling pathway linking cardiac lymphatics, the heart's outer lining (epicardium), and key growth genes that together help regulate cardiac growth.
The study builds on earlier discoveries from the Oliver laboratory that showed lymphatic vessels secrete a protein called Reelin that promotes heart growth by promoting cardiomyocyte proliferation, or when heart muscle cells divide to create new cells. The current study shows Reelin's influence extends beyond heart muscle cells, helping maintain the epicardium, a thin layer of tissue surrounding the heart that is essential for normal cardiac development.
The team found that hearts lacking lymphatic-derived Reelin developed significant defects in the epicardium and loss of some specific populations of fibroblasts - cells that help build and support the heart's structure. The investigators also observed reduced numbers of epicardial cells expressing two critical cardiac factors, the transcription factor WT1 and the growth factor insulin-like growth factor-1 (IGF-1).
"We recently identified the cardiac lymphatic vasculature as an unexpected regulator of heart size during embryonic development," said Oliver, who is also a member of the Robert H. Lurie Comprehensive Cancer Center of Northwestern. "Here, by combining single-cell RNA sequencing with loss-of-function mouse models, we uncover another novel and unexpected critical regulatory network also participating in this process."
The findings may help answer one of developmental biology's most fundamental questions: how organs achieve their correct size.
"When you don't have Reelin, the epicardium is defective," Oliver said. "We know that normally Reelin controls cardiomyocyte proliferation, but now we also know that it is essential for the maintenance and expansion of other cardiac cell types, including the epicardium and fibroblasts, and the expression of WT1 and IGF-1, which are responsible for the growth of the heart."
To explore how Reelin acts on human tissue, the investigators turned to laboratory-grown human "epicardioids," three-dimensional organoids that mimic features of the developing heart. Treating these mini-hearts with Reelin protein increased the expression of multiple epicardial markers, indicating that it promoted epicardial cell fate, supporting the idea that Reelin directly influences epicardial maintenance during development and induces epicardial fate in the adult heart.
The findings suggest that lymphatics' beneficial effect during adult cardiac injury may be partially mediated by Reelin reactivating the dormant epicardium, leading to more efficient repair, Oliver said.
"Reelin expression in cardiac lymphatics declines progressively after birth and becomes nearly undetectable by day seven," Oliver said. "This postnatal decrease coincides with the cessation of cardiac regeneration in newborn mice, suggesting that loss of Reelin may contribute to the arrest of these processes."
Oliver and his research group now suspect lymphatic vessels may function as organ-wide monitoring systems that help determine when growth is complete.
"What we propose is that it's possible that lymphatics function as organ-level quality control systems," Oliver said. "The lymphatics, as they grow into a developing organ and monitor functional features such as interstitial fluid pressure, waste accumulation and tissue stiffness, are saying, 'Okay, everything is looking good; keep growing, keep proliferating. Then, at some point that's good enough, stop there.'"
The study was supported by National Institutes of Health grants RO1HL151388, RO1HL162800, R35GM158171, R01EY032609 and the Brightfocus Foundation new investigator award M2021018N.
