IL4Rα signaling promotes neonatal cardiac regeneration and cardiomyocyte cell cycle activity
Introduction
After myocardial infarction (MI) an adult loses an average of 1 billion cardiomyocytes (CMs) and the damaged myocardium is replaced by stiff, fibrotic tissue [1]. While advances in technology and treatments have improved post-MI survival, endogenous healing mechanisms are inadequate to fully repair the loss of functional CMs and restore cardiac function. Investigating molecular mechanisms that facilitate complete cardiac regeneration in pro-regenerative organisms such as zebrafish, newts, and neonatal mice provides a unique opportunity to discover pathways that can be re-activated in adult mammals to improve post-MI outcomes. Within the first few days of life, neonatal mice fully regenerate their hearts after injury which is accomplished by proliferation of pre-existing CMs, as opposed to contribution from a progenitor or stem cell population [2]. By the end of the first postnatal week, majority of CMs transition to become bi- or multi-nucleated and lose the ability to proliferate [3]. While the adult mammalian heart is incapable of robust CM proliferation, emerging evidence has shown that a subset of adult CMs, specifically mononuclear and diploid CMs (MNDCMs), retain limited proliferative potential [4]. However, the rate of CM proliferation occurs at a rate far too low to functionally recover after MI [[4], [5], [6], [7]]. A potentially promising mechanism to achieve myocardial repair is to identify the pathways promoting the various steps of CM proliferation in pro-regenerative models, such as the neonatal mouse, and reactivate those pathways in the adult heart post injury.
Our lab previously reported that Interleukin 13 (IL13) promotes neonatal mouse CM cell cycle activity and heart regeneration [8]. IL13 is an anti-inflammatory cytokine that shares the common type II IL4Rα/IL13Rα1 receptor heterodimer with Interleukin 4 (IL4). While IL4 and IL13 are well known to contribute to tissue repair via macrophage polarization [[9], [10], [11]], the type II receptor is also expressed on many non-hematopoietic cell types. Its activation by IL4 or IL13 has been known to promote cell cycle activity during both pathogenesis and regenerative wound healing in other organs [12,13]. For example, type II signaling on resident satellite cells is required for clearance of muscle debris and skeletal muscle regeneration [13]. In the lungs, type II receptor activation by IL13 induces mucus cell metaplasia and smooth muscle proliferation, both of which contribute to asthma pathogenesis [12]. These examples demonstrate a profound proliferative role for type II signaling on non-hematopoietic cells. While our prior studies in neonatal mice demonstrated that global depletion of IL13 impairs cardiac regeneration in neonatal mice, the cell types mediating this regenerative phenotype have not been elucidated. Here, we hypothesize that expression and activation of the type II IL13 receptor, IL4Rα/IL13Rα1, specifically on CMs mediates CM cell cycle activity and neonatal heart regeneration.
In the present study, we leverage IL4Rα genetic depletion mouse models to facilitate ablation of the type II receptor, as both IL4Rα and IL13Rα1 receptor subunits are required for signal transduction. We first demonstrate that global IL4Rα deletion (IL4Rα−/−) decreases CM cell cycle activity and cytokinetic completion, which correlates with impaired cardiac regeneration in neonatal mice. Furthermore, knockout of IL4Rα specifically on CMs (IL4Rαfl/fl; Myh6CRE) significantly diminishes CM cell cycle activity and neonatal cardiac regeneration. This demonstrates for the first time that IL4Rα expression directly on CMs promotes pro-proliferative and pro-regenerative responses in a cell autonomous manner. Importantly, activation of the type II receptor by exogenous delivery of IL13 significantly enhances CM DNA synthesis and karyokinesis, while improving overall cardiac healing following MI in non-regenerative mice, suggesting that activation of the type II receptor can improve outcomes past the neonatal regenerative window. Collectively, these data demonstrate a novel role for IL4Rα expression in promoting CM cell cycle activity, including completion of cytokinesis, during cardiac regeneration in neonatal mice and can extend cell cycle activity through karyokinesis past the window of regenerative competence.
Section snippets
Animals
Mice were housed in the Biomedical Resource Center at the Medical College of Wisconsin, an AAALAC-approved facility. All protocols in this study were approved by the local Animal Care and Use Committee. For all in vivo animal experiments, data from male and female mice were pooled. IL4Rα−/− (BALB/c-Il4ratm1Sz/J) mice were purchased from Jackson Laboratories (#003514) and backcrossed with BALB/C mice (Jackson Laboratory). Heterozygous offspring were set up as breeder pairs to obtain IL4Rα−/− and
IL4Rα genetic depletion decreases CM cell cycle activity during early postnatal development
Our previous studies concluded that global depletion of IL13 disrupted early postnatal CM cell cycle activity, which correlated with a lack of neonatal cardiac regeneration. We hypothesized that IL13 signals through the type II receptor to promote CM cell cycle activity and cardiac regeneration; therefore, genetic depletion of IL4Rα, a subunit of the type II IL13 receptor, will phenocopy IL13−/− mice. Cultured CMs from wildtype mice robustly respond to IL13 as illustrated by phosphorylation of
Discussion
Our prior work identified a novel and critical role for IL13 signaling in neonatal cardiac regeneration. IL13 signals predominately through the type II IL4Rα/IL13Rα1 receptor, which has been shown to mediate organ regeneration via signaling in non-hematopoietic cells [12,13]. Here, we demonstrate for the first time that type II receptor signaling, specifically IL4Rα expression, is also critical for cardiac repair in neonatal models. In response to P1 cardiac injury, IL4Rα−/− mice displayed
Grants
This work was supported by National Institute for Health [T32HL7852 and F31HL150919 to S.J.P, R01HL141159 to C.C.O., R01HL155085 to MP, T32HL134643 and F32HL150958 to M.A.F.,] and by the Cardiovascular Center's A.O. Smith [M.A.F.]. Work was supported by American Heart Association [18CDA34110204 to M.P.].
Declaration of Competing Interest
None declared.
Acknowledgements
We thank Tina Wan and Dr. John Auchampach and the National Institute of Health (S10OD025038 to John Auchampach) for support with echocardiography. We thank Dylan Wodsedalek for excellent technical support. We thank Dr. Yi Guang Chen and Dr. Ashley Ciecko for flow cytometry consultation, and Dr. Frank Brombacher for graciously providing IL4Rαfl/fl mice.
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