Embryonic microRNA Labeled as Fountain of Youth for Heart Cells

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Scientists at the Lewis Katz School of Medicine at Temple University (LKSOM) report that it may be possible to reverse heart damage and restore heart function after disease, even after a severe heart attack. The study (Transient Introduction of miR-294 in the Heart Promotes Cardiomyocyte Cell Cycle Reentry After Injury), published in Circulation Research, is the first to show that a small RNA molecule known as miR-294, when introduced into heart cells, can reactivate heart cell proliferation and improve heart function in mice that have suffered the equivalent of a heart attack in humans, according to the researchers.

“Embryonic heart is characterized of rapidly dividing cardiomyocytes required to build a working myocardium. Cardiomyocytes retain some proliferative capacity in the neonates but lose it in adulthood. Consequently, a number of signaling hubs including microRNAs are altered during cardiac development that adversely impacts regenerative potential of cardiac tissue. Embryonic stem cell cycle miRs are a class of microRNAs exclusively expressed during developmental stages; however, their effect on cardiomyocyte proliferation and heart function in adult myocardium has not been studied previously,” the investigators wrote.

“[We wanted to] determine whether transient reintroduction of embryonic stem cell cycle miR-294 promotes cardiomyocyte cell cycle reentry enhancing cardiac repair after myocardial injury. miR-294 is expressed in the heart during development, prenatal stages, lost in the neonate, and adult heart confirmed by qRT-PCR and in situ hybridization. Neonatal ventricular myocytes treated with miR-294 showed elevated expression of Ki67, p-histone H3, and Aurora B confirmed by immunocytochemistry compared with control cells. miR-294 enhanced oxidative phosphorylation and glycolysis in Neonatal ventricular myocytes measured by seahorse assay. Mechanistically, miR-294 represses Wee1 leading to increased activity of the cyclin B1/CDK1 complex confirmed by qRT-PCR and immunoblot analysis.

“Next, a doxycycline-inducible AAV9-miR-294 vector was delivered to mice for activating miR-294 in myocytes for 14 days continuously after myocardial infarction. miR-294–treated mice significantly improved left ventricular functions together with decreased infarct size and apoptosis eight weeks after MI. Myocyte cell cycle reentry increased in miR-294 hearts analyzed by Ki67, pH3, and AurB (Aurora B kinase) expression parallel to increased small myocyte number in the heart. Isolated adult myocytes from miR-294 hearts showed increased 5-ethynyl-2′-deoxyuridine+ cells and upregulation of cell cycle markers and miR-294 targets eight weeks after MI.

“[We concluded that] ectopic transient expression of miR-294 recapitulates developmental signaling and phenotype in cardiomyocytes promoting cell cycle reentry that leads to augmented cardiac function in mice after myocardial infarction.”

“In previous work, we discovered that miR-294 actively regulates the cell cycle in the developing heart,” said Mohsin Khan, PhD, assistant professor of physiology at the Center for Metabolic Disease Research at LKSOM. “But shortly after birth miR-294 is no longer expressed.”

Khan and colleague Raj Kishore, PhD, professor of pharmacology and medicine and director of the Stem Cell Therapy Program in the Center for Translational Medicine at LKSOM, both senior investigators on the new study, wondered whether miR-294 could serve as a sort of fountain of youth for heart cells.

“The heart is very proliferative when miR-294 is expressed in early life,” Kishore explained. “We wanted to see if reintroducing it into adult heart cells would turn them back to an embryonic-like state, allowing them to make new heart cells.”

The researchers tested their idea in mice that had myocardial infarction. Mice were treated with miR-294 continuously for two weeks after sustaining myocardial injury. Two months following treatment, the researchers observed noticeable improvements in heart function and a decrease in the area of damaged tissue. Examination of treated heart cells revealed evidence of cell cycle reentry, indicating that the cells had been reactivated, regaining the ability to produce new cells.

“The miR-294 treatment reawakened an embryonic signaling program in the adult heart cells,” said Khan. “Because of this, the old heart cells were no longer quite like adult cells, but neither were they fully embryonic. In this in-between state, however, they had the ability to make new cells.”

The researchers were able to control miR-294 expression, turning it on or off and thereby dictating the amount of proliferative activity in the heart.

Khan and Kishore plan next to replicate the study in a large animal model. They also want to gain a deeper understanding of what miR-294 is doing in the heart. “There is evidence that it does more than control the cell cycle,” explained Khan. “If it has multiple targets, we need to find them.”

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