Assessment of embryonic myocardial cell differentiation using a dual fluorescent reporter system

Recent studies have identified the existence of undifferentiated myocardial cells during early embryonic as well as post-natal stages of heart development. While primitive cells present in the precardiac mesoderm can differentiate into multiple cell types of the cardiovascular system, the developmental potential of undifferentiated cells identified in the ventricular myocardium after chamber formation is not well characterized. A deeper understanding of mechanisms regulating myocardial cell differentiation will provide further insights into the normal and pathological aspects of heart development. Here, we showed that Nkx2.5 positive and sarcomeric myosin negative cells were predominantly localized in the right ventricular myocardium of CD1 mice at E11.5 stage. We confirmed that myocardial regions negative for saromeric myosin were also devoid of atrial natriuretic factor (ANF). These observations are consistent with our previous study, which showed that ANF expression is restricted to moderately differentiated and mature myocardial cells in E11.5 myocardium of C3H/FeJ mice. Further, we found that the receptor c-Kit, a marker for early embryonic myocardial progenitor cells, is not expressed in the undifferentiated cells of the E11.5 myocardium. To monitor the differentiation potential of Nkx2.5(+)/ANF(-) cells in vitro, we developed a novel double fluorescent reporter system. Subsequently, we confirmed that the majority of Nkx2.5(+)/ANF(-) cells expressed mature myocyte markers such as sarcomeric myosin, MLC2V and alpha-cardiac actin after 48 hrs in culture, albeit at lower levels compared to Nkx2.5(+)/ANF(+) or Nkx2.5(-)/ANF(+) cell populations. Our results suggest that fluorescent reporters under the control of lineage-specific promoters can be used to study myocardial cell differentiation in response to various exogenous or pharmacological agents.