We propose that dynamic and sustained RA production by the zebrafish endocardium, in addition to the epicardium, is an important component of the capacity to achieve local regenerative cardiogenesis. Indeed, we show it is a feature of a second non-mammalian vertebrate that we find also to be capable of injury-induced CM proliferation, Polypterus senegalus. The non-regenerative mouse heart does increase RA synthesis in response to injury; however, the cell types, prominence, and kinetics of RA production in response to myocardial damage are quite different (). These differences implicate the endocardium and non-myocardial RA production as targets in attempts to increase regenerative capacity in the injured mammalian heart.
The molecular mechanisms by which the endocardium is activated and induces raldh2
will require further clarification. Clues may come from the fact that we could stimulate raldh2
induction by LPS injection, and that injury triggered organ-wide morphologic changes in the endocardium. One possibility is that growth factors or cytokines are released after injury and impact the permeability of endocardial cells (Nagy et al., 2008
), with raldh2
induction being a response to this change. Indeed, Heg is required for vascular permeability in mice (Kleaveland et al., 2009
), and heg
is shown here to be a robust early marker of the activated endocardium in zebrafish. To date, we have not been able to induce a strong, organ-wide response by injection of individual murine pro-inflammatory factors such as IL-6, IL-1β, IFNγ, and TNFαK.K. and K.D.P., unpublished results).
It has been known for several years that RA signaling is critical for myocardial proliferation during embryonic heart development. While it was initially suspected that embryonic ventricular compact CMs receive epicardial RA and are instructed to proliferate, a number of experiments argue against this mechanism. These include the fact that RXRα function was not required in CMs for normal ventricular cardiogenesis (Chen et al., 1998
; Merki et al., 2005
; Subbarayan et al., 2000
; Tran and Sucov, 1998
), and that RA was insufficient on its own as a mitogen in cultured cardiac slices (Stuckmann et al., 2003
). Our findings appear to parallel these studies, given that RA presence and reception were required for regenerative proliferation, yet we could not influence CM proliferation by introducing RAR ligand. It is also possible that RA production within or distant from areas of injury has multiple functions germane to different cardiac cell types and heart regeneration, a relevant issue during embryonic cardiac growth (Brade et al., 2011
; Jenkins et al., 2005
). As mechanisms by which RA influences cardiac proliferation and patterning in embryos are uncovered, these findings are likely to provide insight into the influences of endocardial and epicardial RA during heart regeneration.