CBL66 Sigma-AldrichAnti-Atrial Natriuretic Peptide Antibody, clone A6

Interventricular septum myocardium was studied in 40 patients with obstructive hypertrophic cardiomyopathy. Immunohistochemical assay revealed c-kit-positive resident cardiac stem cells in 82.5% patients. The content of the connective tissue and myofibrillar disarray zones and the degree of cardiomyocyte hypertrophy and myolysis were determined. In 30% cases, cardiomyocytes containing atrial natriuretic peptide were detected in the interventricular septum myocardium. The data were compared with clinical and functional parameters of patients. It was found that cardiac stem cells are present in patients, whose myocardium was characterized by increased density of the connective tissue, hypertrophy of mature cardiomyocytes, medium degree of myolysis in them, and accumulation of natriuretic peptide, a cardiac failure marker, in cardiomyocytes.

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.

Human mesenchymal stem cells (hMSCs) have only a limited differentiation potential toward cardiomyocytes. Forced expression of the cardiomyogenic transcription factor myocardin may stimulate hMSCs to acquire a cardiomyogenic phenotype, thereby improving their possible therapeutic potential. hMSCs were transduced with green fluorescent protein (GFP) and myocardin (hMSC(myoc)) or GFP and empty vector (hMSC). After coronary ligation in immune-compromised NOD/scid mice, hMSC(myoc) (n = 10), hMSC (n = 10), or medium only (n = 12) was injected into the infarct area. Sham-operated mice (n = 12) were used to determine baseline characteristics. Left ventricular (LV) volumes and ejection fraction (EF) were serially (days 2 and 14) assessed using 9.4-T magnetic resonance imaging. LV pressure-volume measurements were performed at day 15, followed by histological evaluation. At day 2, no differences in infarct size, LV volumes, or EF were observed among the myocardial infarction groups. At day 14, left ventricular ejection fraction in both cell-treated groups was preserved compared with the nontreated group; in addition, hMSC(myoc) injection also reduced LV volumes compared with medium injection (p < .05). Furthermore, pressure-volume measurements revealed a significantly better LV function after hMSC(myoc) injection compared with hMSC treatment. Immunohistochemistry at day 15 demonstrated that the engraftment rate was higher in the hMSC(myoc) group compared with the hMSC group (p < .05). Furthermore, these cells expressed a number of cardiomyocyte-specific markers not observed in the hMSC group. After myocardial infarction, injection of hMSC(myoc) improved LV function and limited LV remodeling, effects not observed after injection of hMSC. Furthermore, forced myocardin expression improved engraftment and induced a cardiomyocyte-like phenotype hMSC differentiation.

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 ( approximately 20%) and E18.5 ( approximately 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.