Macrophages Help Newborn Mice Heal Their Hearts

In 1974, researchers observed that the hearts of salamanders heal fully after an injury, providing one of the first accounts of cardiac regeneration.¹ A few decades later, scientists showed that zebrafish and axolotls could regenerate their wounded hearts.^2,3

While these animals retain the regenerative ability throughout adulthood, most mammals, including mice and humans, lose this capacity shortly after birth.⁴ Scientists are interested in determining why adults cannot heal their hearts after a heart attack without forming scar tissue.

Previous research by some groups revealed that macrophages play an important role in the regeneration of newborn hearts.⁵ “We generally had an idea that newborn macrophages versus adult macrophages were different,” said Connor Lantz, a bioinformatician at Northwestern University. “But I don't think we had any idea about what the molecular players were behind this.”

Now, Lantz and his team have shown that macrophages in newborn mice hearts eliminate dying cells, triggering a signaling cascade that results in the proliferation of heart muscle cells, allowing damaged heart muscle to regenerate.⁶ This response was absent in adult mice. The results, published in Immunity, highlight an immunometabolic mechanism that influences age-dependent cardiac regeneration and offer a potential therapeutic target to improve cardiac tissue repair in adults.

“Their results are very exciting,” said Nadia Rosenthal, a cardiac development and regeneration researcher at The Jackson Laboratory, who was not involved in the study. “[The results] allow you to start to pinpoint exactly where the real changes are made from the neonate to the adult.”

To investigate the age-dependent response after cardiac injury, Lantz and his team surgically tied the coronary arteries in one-day-old newborns and eight-week-old adult mice. While neutrophils, monocytes, and macrophages accumulated in adult hearts, only macrophages gathered in the newborn hearts.

Single-cell RNA sequencing revealed that macrophage populations gathered in adult and newborn hearts after injury were different: Macrophages in newborns expressed genes implicated in cardiac regeneration and development, while those in adults expressed cell migration- and fibroblast proliferation-associated genes.

Lantz and his team analyzed the intercellular communication networks using a bioinformatic tool to compare macrophage signaling in newborn and adult hearts.⁷ Compared to adults, newborn hearts showed increased signaling of proteins associated with efferocytosis, or engulfing of dying cells by macrophages.

To investigate whether efferocytosis influences cardiac regeneration, Lantz and his team deleted the myeloid-epithelial-reproductive tyrosine kinase (MerTK) receptor on macrophages that mediates efferocytosis by recognizing dying cells. Three weeks after cardiac injury, wild type newborn mice that could carry out efferocytosis completely recovered their heart function with little scarring. In contrast, newborn mice lacking Mertk exhibited scarring and reduced heart function.

Red-colored cells with some blue and some green nuclei. — Newborn cardiomyocytes (red) proliferated (green nuclei) in response to secretions from macrophages that engulfed dying cells. Nuclei in non-proliferating cells are depicted in blue.
Connor Lantz

Mass spectrometry revealed that compared to Mertk deficient hearts, macrophages from wild type neonatal hearts exhibited increased fatty acid metabolism. Specifically, Lantz and his team observed increased amounts of thromboxane A2, a product of arachidonic acid metabolism, after cardiac injury. In contrast, thromboxane A2 levels reduced in macrophages from hearts of newborn mice lacking Mertk and adult mice post injury, indicating an age and MerTK-dependent arachidonic acid metabolism in hearts.

Cultured heart muscle cells, or cardiomyocytes, treated with a thromboxane A2-mimicking molecule showed increased proliferation, suggesting that macrophage-derived thromboxane A2 induces cardiomyocyte proliferation.

Treating cardiomyocytes with the supernatant obtained from macrophages after efferocytosis, containing molecules secreted by macrophages, induced their proliferation. Macrophages deficient in thromboxane A2 did not cause this effect, validating that thromboxane A2 secreted by newborn macrophages after they engulf dead cells triggers cardiomyocyte proliferation.

“This was a surprise,” said Lantz, who did not expect bioactive lipid metabolism to be involved in newborn cardiomyocyte regeneration. “It was kind of cool.”

Finally, the researchers sought to understand the mechanism by which thromboxane A2 acts on cardiomyocytes. They focused on a known regulator of cardiomyocyte proliferation in newborn hearts called HIPPO-yes-associated protein (YAP).⁸ They observed that cardiomyocytes bordering the cardiac injury in newborn hearts accumulated YAP. The protein also concentrated in cultured cardiomyocytes treated with thromboxane A2-mimetic, hinting at a molecular mechanism where macrophages secrete thromboxane A2 after efferocytosis in injured hearts to modulate neighboring cardiomyocyte proliferation through YAP signaling.

“This is super exciting because it gives you more insight into the way in which heart regeneration could be reactivated, perhaps in adults,” said Rosenthal. She added that unlike mice, humans express two isoforms of thromboxane A2, but the results point researchers in the right direction to manipulate cardiac tissue repair.

Rosenthal noted that previous studies by her and others have found that genetically diverse mice have distinct responses to heart attacks similar to humans responding differently, suggesting that more research is needed to see how the results translate to humans.

Using publicly available datasets, Lantz found that human hearts express MERTK and the gene encoding the enzyme that synthesizes thromboxane A2. “So, I think there is translatable evidence there.” But the big picture is more complex, he noted. “We focus on this one pathway that is necessary for regeneration, but there's a bunch of other things that also need to be occurring for complete regeneration.”

Aside from application in designing therapeutics, both Lantz and Rosenthal said that the results raise important questions about why this signaling is lost soon after birth. “There's something, as we age…that causes a change, and now this leads [the macrophages] to be more proinflammatory,” said Lantz. “I think that’s the next question. Trying to figure out why.”