The adult heart has a very limited ability to repair and regenerate itself following myocardial infarction and with significant damage, there is a severe loss of cardiomyocytes. The intrinsic inability to replace these cardiomyocytes leads to heart failure. Enhancing the heart?s intrinsic reparative and regenerative mechanisms to generate new cardiomyocytes has clear therapeutic benefits. One therapeutic strategy would be to utilize stem cells that are resident in the heart. One potential resident stem cell is the c-Kit(+) cell. These in situ c- Kit(+) cells have been reported to be an important endogenous source of new cardiomyocytes in vivo; however, c-Kit lineage tracing experiments have given contradictory results in this regard. Nonetheless, it remains an open question as to whether the contribution of c-Kit(+) cells can be significantly augmented biologically or pharmacologically. Recently, we have discovered that the Wnt regulator Sfrp2 promotes cardiac regeneration via the differentiation of c-Kit(+) cells into cardiomyocytes. Our data shows that Sfrp2 promotes c-Kit(+) cell differentiation in vivo. This correlated with significant improvements in cardiac repair and function following MI. Importantly, ablation of c-Kit(+) cells prevented Sfrp2 mediated cardiac regeneration. Moreover, our data suggests that Sfrp2 promotes c-Kit(+) differentiation into cardiomyocytes via ?-catenin inhibition mediated by Wnt11 and Fzd5. Accordingly, we hypothesize that Sfrp2 is a key regulator of cardiac regeneration via the activation of c-Kit(+) cell differentiation into cardiomyocytes. We propose three Specific Aims.
In Aim 1 we will examine the role of ?-catenin inhibition in mediating c-Kit(+) cell differentiation into cardiomyocytes using ?-catenin gain-of-function and lack-of-function approaches both in vitro and in vivo. In vivo, we will inject c-Kit(+) cells, engineered for ?-catenin gain-of-function/loss-of-function approaches, into the infarcted heart and track their fate.
In Aim 2 we will provide definitive evidence for the role of ?-catenin in mediating the response of Sfrp2 in vivo by using genetic knockout models. These genetic models will either completely block ?-catenin activity or render ?-catenin permanently active specifically in c-Kit(+) cells. We will then lineage trace these modified in situ c-Kit(+) cells in response to MI and Sfrp2.
In Aim 3 we will examine our hypothesis that Sfrp2 binds to canonical Wnt3a leaving non-canonical Wnt11 to bind and activate Fzd5; thereby activating a non-canonical pathway to inhibit ?-catenin. Binding and competition assays will verify that Wnt3a and Wnt11 bind to Fzd5. Wnt blocking antibodies will show that sequestration of Wnt3a will promote c- Kit(+) differentiation via Wnt11 as proof-of-concept. This study will be repeated with Sfrp2 in place of blocking antibodies. Fluorescence assays will be employed to show Wnt11/Fzd5 mediated inactivation of ?-catenin involves caspases. Genetic knockout models and lineage tracing will validate the importance of Fzd5 and Wnt11 for c-Kit(+) differentiation into cardiomyocytes in vivo.
The adult heart has a very limited ability to repair and regenerate following myocardial infarction. Cardiomyocyte loss is severe but the intrinsic ability to replace them is limited; leading to eventual heart failure. Enhancing the heart?s innate reparative and regenerative mechanisms to generate new cardiomyocytes has clear therapeutic benefits. This could be achieved through the activation of resident stem cells. One putative heart stem cell is the c-Kit(+) cell. The role of endogenous c-Kit(+) in cardiac regeneration is controversial; however, it is an open question as to whether the contribution of these cells can be significantly augmented biologically or pharmacologically. Recently, we have discovered that the Wnt regulator Sfrp2 promotes cardiac regeneration via the differentiation of c-Kit(+) cells into cardiomyocytes. In this project we will define the molecular mechanisms underlying c-Kit(+) differentiation into cardiomyocytes in response to Sfrp2. These studies should open new avenues for the development of novel strategies to regenerate the heart following myocardial injury.
| Hodgkinson, Conrad P; Gomez, José A; Baksh, Syeda Samara et al. (2018) Insights from molecular signature of in vivo cardiac c-Kit(+) cells following cardiac injury and ?-catenin inhibition. J Mol Cell Cardiol 123:64-74 |
