Defining cardiac cell populations and relative cellular composition of the early fetal human heart.

While the adult human heart is primarily composed of cardiomyocytes, fibroblasts, endothelial and smooth muscle cells, the cellular composition during early development remains largely unknown. Reliable identification of fetal cardiac cell types using protein markers is critical to understand cardiac development and delineate the cellular composition of the developing human heart. This is the first study to use immunohistochemistry (IHC), flow cytometry and RT-PCR analyses to investigate the expression and specificity of commonly used cardiac cell markers in the early human fetal heart (8-12 post-conception weeks). The expression of previously reported protein markers for the detection of cardiomyocytes (Myosin Heavy Chain (MHC) and cardiac troponin I (cTnI), fibroblasts (DDR2, THY1, Vimentin), endothelial cells (CD31) and smooth muscle cells (α-SMA) were assessed. Two distinct populations of cTnI positive cells were identified through flow cytometry, with MHC positive cardiomyocytes showing high cTnI expression (cTnIHigh) while MHC negative non-myocytes showed lower cTnI expression (cTnILow). cTnI expression in non-myocytes was further confirmed by IHC and RT-PCR analyses, suggesting troponins are not cardiomyocyte-specific and may play distinct roles in non-muscle cells during early development. Vimentin (VIM) was expressed in cultured ventricular fibroblast populations and flow cytometry revealed VIMHigh and VIMLow cell populations in the fetal heart. MHC positive cardiomyocytes were VIMLow whilst CD31 positive endothelial cells were VIMHigh. Using markers investigated within this study, we characterised fetal human cardiac populations and estimate that 75-80% of fetal cardiac cells are cardiomyocytes and are MHC+/cTnIHigh/VIMLow, whilst non-myocytes comprise 20-25% of total cells and are MHC-/cTnILow/VIMHigh, with CD31+ endothelial cells comprising ~9% of this population. These findings show distinct differences from those reported for adult heart.

Expression and localisation of thymosin beta-4 in the developing human early fetal heart.

BACKGROUND: The objective of this study was to investigate the expression and localisation of thymosin ß4 (Tß4) in the developing human heart. Tß4 is a cardioprotective protein which may have therapeutic potential. While Tß4 is an endogenously produced protein with known importance during development, its role within the developing human heart is not fully understood. Elucidating the localisation of Tß4 within the developing heart will help in understanding its role during cardiac development and is crucial for understanding its potential for cardioprotection and repair in the adult heart. METHODS: Expression of Tß4 mRNA in the early fetal human heart was assessed by PCR using both ventricular and atrial tissue. Fluorescence immunohistochemistry was used to assess the localisation of Tß4 in sections of early fetal human heart. Co-staining with CD31, an endothelial cell marker, and with myosin heavy chain, a cardiomyocyte marker, was used to determine whether Tß4 is localised to these cell types within the early fetal human heart. RESULTS: Tß4 mRNA was found to be expressed in both the atria and the ventricles of the early fetal human heart. Tß4 protein was found to be primarily localised to CD31-expressing endothelial cells and the endocardium as well as being present in the epicardium. Tß4-associated fluorescence was greater in the compact layer of the myocardial wall and the interventricular septum than in the trabecular layer of the myocardium. CONCLUSIONS: The data presented illustrates expression of Tß4 in the developing human heart and demonstrates for the first time that Tß4 in the human heart is primarily localised to endothelial cells of the cardiac microvasculature and coronary vessels as-well as to the endothelial-like cells of the endocardium and to the epicardium.