Abstract
Phenylethanolamine n-methyltransferase (Pnmt) catalyzes the conversion of norepinephrine to epinephrine, and thus serves as a marker for adrenergic cells. We employed a combination of immunofluorescent histochemical staining and genetic fate-mapping strategies to show that two separate Pnmt+ cell populations contribute to heart development. Intrinsic cardiac adrenergic (ICA) cells originate from the primary heart field, and contribute to pacemaking, conduction, and working (contractile) myocardium. A second population of cardiac Pnmt+ cells is derived from migrating neural crest. These neural crest adrenergic (NCA) cells appear to contribute to cardiac neurons. By adulthood, most of the Pnmt+ cells show a distinctively left-sided orientation in the heart, with nearly 90% of them being found in the left atrium and ventricle. Surprisingly large swaths of ventricular muscle are derived from Pnmt+ primer cells. Since this region of the heart is highly vulnerable to coronary artery disease and often sustains varying degrees of damage following myocardial infarction, we hypothesize that directed stem cell differentiation into Pnmt+ primer cells could serve as a valuable resource for repair and/or regeneration of left ventricular myocardium for heart disease patients. To test this hypothesis, we have generated stable recombinant mouse embryonic stem cell (mESC) lines that express various fluorescent marker proteins under the control of the endogenous Pnmt gene regulatory network. These cells can be rapidly expanded in culture, sorted, and used for transplantation studies in animal models to determine their therapeutic effectiveness. The cells can be induced along cardiogenic or neurogenic pathways in vitro, and the resulting Pnmt+ cells from each population can then be collected and tested in vivo. To achieve this goal, we have knocked-in a nuclear-localized enhanced green fluorescent protein into the Pnmt locus to create Pnmt-nEGFP recombinant mESCs and mice. We show that nEGFP expression is specifically expressed in Pnmt+ cells in vitro and in vivo. This strategy allows us to identify and isolate Pnmt+ cells to evaluate their effectiveness for cardiac regenerative medicine applications. .
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