Neuregulin-1, in a Conducive Milieu with Wnt/BMP/Retinoic Acid, Prolongs the Epicardial-Mediated Cardiac Regeneration Capacity of Neonatal Heart Explants

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.

Regeneration of the heart: from molecular mechanisms to clinical therapeutics

“…VEGF mRNA has been administrated to patients via direct intramyocardial injection, showing that it may be safe for introducing genetic material to the cardiac muscle [ 178 ]. Nrg1 sustains the epicardial-mediated cardiac regeneration capacity of neonatal heart explants [ 179 ]. Oxytocin also activates epicardial cells and promotes heart regeneration after cardiac injury [ 180 ].…”

Comparative Analysis of Heart Regeneration: Searching for the Key to Heal the Heart—Part II: Molecular Mechanisms of Cardiac Regeneration

“…They observed that the Tp53 network is transiently suppressed during zebrafish heart regeneration, increasing CMs proliferation, indicating that this molecular pathway is directly related to innate regeneration in zebrafish [ 141 ]. In addition, zebrafish ex vivo experiments performed by Arora et al [ 142 ] demonstrated that the combination of Wnt family member 3 (Wnt3), Bone morphogenetic protein 4 (BMP4) and Nrg1 has a regenerative effect in damaged neonatal hearts in culture. The combination of these growth factors promotes cardiac repair via epicardial cell activation, their proliferation and migration to the injury site, followed by CMs recruitment [ 142 ] ( Figure 3 A).…”

“…In addition, zebrafish ex vivo experiments performed by Arora et al [ 142 ] demonstrated that the combination of Wnt family member 3 (Wnt3), Bone morphogenetic protein 4 (BMP4) and Nrg1 has a regenerative effect in damaged neonatal hearts in culture. The combination of these growth factors promotes cardiac repair via epicardial cell activation, their proliferation and migration to the injury site, followed by CMs recruitment [ 142 ] ( Figure 3 A). Finally, Nrg1 regulates CMs proliferation through the activation of the Ras/Mapk pathway, which is controlled by feedback attenuator Dual specificity phosphatase 6 (Dusp6), an ERK phosphatase [ 143 ] ( Figure 3 A).…”

Neuregulin-1, a potential therapeutic target for cardiac repair

“…As a cardiac regeneration growth factor, NRG1 has attracted the attention of cardiovascular medicine. NRG1 has been shown to not only stimulate cardiomyocyte proliferation ( Zhao et al, 1998 ; Bersell et al, 2009 ; D'Uva et al, 2015 ), but also act within angiogenesis, extracellular matrix remodeling, cardiomyocyte proliferation, stem-cell recruitment, and others, that improve cardiac function ( Mendes-Ferreira et al, 2016 ; Rebouças et al, 2016 ; Arora et al, 2021 ). Together, these mechanisms promote myocardial repair and improvement of the cardiac function, that providing a novel molecular strategy targeting at regenerating the injured myocardium.…”