Abstract
Rationale: Cardiac sympathetic nerves are required for endogenous repair of the mammalian neonatal heart in vivo, but the underlying mechanism is unclear. Objective: We tested the hypothesis that a combination of cardiac developmental growth factors Wnt3a, BMP4 and Neuregulin (NRG-1), compensate for denervation and support cardiac regeneration in explanted neonatal mammalian hearts. Methods and Results: Hearts from 2-day old neonatal mice were harvested, lesioned at the apex and grown ex vivo for 21 days under defined conditions. Hearts grown in canonical cardiomyocyte culture media underwent complete coagulative necrosis, a process resembling ischemic cell death, by day 14. However, the addition of Wnt3a, BMP-4 and NRG-1, maintained cellular integrity and restored the endogenous regenerative program. None of these factors alone, or in any paired combination, were sufficient to induce regeneration in culture. rNRG-1 alone significantly reduced the accumulation of double strand DNA damage at Day 3; (-NRG-1: 60±12%; +NRG-1: 8±3%; P<0.01) and prevented coagulative necrosis at Day 14. Short-term addition of rWnt3a and rBMP-4 (day 0-3, NRG-1+) increased WT1 expression (a marker of epicardial cells) 7-fold, epicardial proliferation (78±17 cells vs. 21±9 cells; P<0.05), migration and recellularization (80±22 vs. zero cells; P<0.01; n=6) at the injury site on day 14. Conclusions: A novel explant culture system maintains three-dimensional neonatal mouse hearts and the mammalian neonatal cardiac regenerative program ex vivo. We identified that rNRG-1, plus short-term activation of Wnt- and BMP-signaling, promotes cardiac repair via epicardial cell activation, their proliferation and migration to the injury site, followed by putative cardiomyocyte recruitment. This novel technique will facilitate future studies of mammalian cardiac regeneration and may be useful in cardiac-specific drug testing.
Highlights
Cardiovascular disease accounts for more deaths than any other disease[1], in large part due to an inability of the adult mammalian heart to sufficiently regenerate lost tissue following injury
Canonical cardiomyocyte culture media used in the maintenance of cardiomyocyte monolayers contains several growth factors together with fetal bovine serum (Base+FBS)
H2AX immunofluorescence showed a significant reduction in DNA damage in rNRG-1 treated explants compared to controls (D, E)
Summary
Cardiovascular disease accounts for more deaths than any other disease[1], in large part due to an inability of the adult mammalian heart to sufficiently regenerate lost tissue following injury. E.g., teleost fish species and urodele amphibians, such as the newt, retain a capacity to regenerate lost cardiac tissue through a process that involves activation of the epicardium and cardiomyocyte proliferation[2,3,4,5]. It was thought that the mammalian heart was incapable of such a response to acute injury. 0.45-1% of human cardiomyocytes are replaced per year throughout life[9]. These findings have propelled a major drive toward determining the mechanisms of endogenous mammalian cardiac regeneration
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