Ultrastructural and immunocharacterization of undifferentiated myocardial cells in the developing mouse heart

Abstract

The recent discovery of several myogenic cardiac progenitor cells in the post-natal heart suggests that some myocardial cells may remain undifferentiated during embryonic development. In this study, we examined the subcellular characteristics of the embryonic (E) mouse ventricular myocardial cells using transmission electron microscopy (TEM). At the ultrastructural level, we identified three different cell populations within the myocardial layer of the E11.5 heart. These cells were designated as undifferentiated cells (43 ± 6%), moderately differentiated cells (43 ± 2%) and mature cardiomyocytes (14 ± 4%). Undifferentiated cells contained a large nucleus and sparse cytoplasm with no myofibrillar bundles. Moderately differentiated cells contained randomly arranged myofilaments in the cytoplasm. In contrast, mature cardiomyocytes contained well-developed sarcomere structures. We also confirmed the presence of similar undifferentiated cells albeit at low levels in the E16.5 (∼20%) and E18.5 (∼7%) myocardium. Further we used immunogold labeling technique to test whether these distinct cell populations were also positive for markers such as Nkx2.5, ISL1 and ANF. A preponderance of anti-Nkx2.5 label was found in the undifferentiated and moderately differentiated cell types. Anti-ANF label was found only in the cytoplasmic compartment of moderately differentiated and mature myocardial cells. All of the undifferentiated cells were negative for anti-ANF labeling. We did not find immuno-gold labeling with ISL1 in any of the three myocardial cell types. Based on these results, we suggest that embryonic myocardial cell differentiation is a gradual process and undifferentiated cells expressing Nkx2.5 in post-chamber myocardium may represent a progenitor cell population while cells expressing Nkx2.5 and ANF represent differentiating myocytes.

Functional characterization of cardiac progenitor cells and their derivatives in the embryonic heart post‐chamber formation

“…known cardiac marker Nkx2.5, ultrathin sections of E11.5 myocardium were processed for immunogold labeling. In agreement with our previous study (Zhang and Pasumarthi, 2007), both cell types expressed Nkx2.5 (Fig. 1C,D).…”

“…1) (Zhang and Pasumarthi, 2007;McMullen et al, 2008). Interestingly, Nkx2.5 þ MLC2v þ ANF þ cells, the predominant cell type identified in this study, comprise $50% of total RV cells and roughly half of them are positive for conduction system markers AChE or Cx40.…”

“…We have previously shown that the number of undifferentiated ventricular cells expressing only Nkx2.5 decreases significantly from E11.5 to E18.5 stage in vivo (Zhang and Pasumarthi, 2007). Since the fate of these cells in vivo could not be studied directly, we designed a triple fluorescent reporter system (NEMEAD) to track the undifferentiated cells in vitro.…”

“…By postnatal ages, the sarcoplasmic reticulum (SR) of cardiomyocytes is fully developed, and the myocyte contraction is regulated by the influx of Ca 2þ as well as release of intracellular Ca 2þ from SR stores through a process known as calcium-induced calnot well established whether any CPCs are left after chamber formation ($E11.5) and whether all myocardial cells at this stage are fully competent for contractile function. Efforts to identify CPCs in the E11.5 heart are limited due to the down-regulation of previously reported CPC markers such as MesP1 and 2, c-Kit and Isl1 (Buckingham et al, 2005;Laugwitz et al, 2005;Wu et al, 2006;Zhang and Pasumarthi, 2007). We have recently shown that E11.5 myocardial cells in C3H/FeJ mouse strain represent a heterogeneous population consisting of both undifferentiated and differentiated cells (Zhang and Pasumarthi, 2007).…”

“…Efforts to identify CPCs in the E11.5 heart are limited due to the down-regulation of previously reported CPC markers such as MesP1 and 2, c-Kit and Isl1 (Buckingham et al, 2005;Laugwitz et al, 2005;Wu et al, 2006;Zhang and Pasumarthi, 2007). We have recently shown that E11.5 myocardial cells in C3H/FeJ mouse strain represent a heterogeneous population consisting of both undifferentiated and differentiated cells (Zhang and Pasumarthi, 2007). We also showed that Nkx2.5 þ and ANF À cells (i.e., putative CPCs) were predominantly localized in the right ventricular (RV) myocardium in vivo (McMullen et al, 2008).…”