Development

How do cardiomyocytes differentiate?

Cardiomyocytes are the chief cell type in the heart and their coordinated contraction as a mass is responsible for the pumping of blood around the developing embryo. Cardiac specification occurs very early on during embryonic development. For instance, in mouse embryos this occurs as early as day 6.5 post fertilization and involves the development of a population of cardiac precursor cells in the lateral posterior epiblast just prior to formation of the primitive streak – a structure which marks the beginning of gastrulation. During gastrulation, as the epiblast cells migrate through the primitive streak, they undergo epithelial-mesenchymal transition and form the mesoderm. Following the induction of mesoderm, the cardiac precursor cells become irreversibly specified into cardiomycytes, and this process of cardiac determination is precisely regulated in time and space by an interplay of various signaling pathways, especially those activated by the Wnt, TGF-beta, FGF, or retinoic acid superfamily of extracellular signaling molecules [1][2]. A stage-specific role of these signaling pathways have been identified through in vivo and in vitro analyses of embryonic stem cells, as seen in the case of Wnt proteins whose activation is essential for the generation of mesodermal germ layer but needs to be subsequently turned off during the differentiation of cardiac progenitor cells from the mesoderm [3][4].

The biochemical signaling pathways involved in cardiomyogenesis are directly or indirectly regulated by a number of mechanical signals throughout cardiac development. A classic example is the complex interplay that takes places between E-cadherin/β-catenin mediated mechanosignaling and Wnt/β-catenin mediated chemical signaling. During development, the nuclear translocation of β-catenin, a transcriptional cofactor in the Wnt signaling pathway, is controlled by the extent of cell-cell contacts formed between cells. Before and during early gastrulation, the epiblast layer assumes a tightly packed cellular morphology due to an increase in E-cadherin mediated mechanical linkages between cells. This leads to a corresponding increase in membrane-bound β-catenin and Wnt signaling. The epiblast responds to Wnt signaling from the underlying endoderm, by release of β-catenin from the membrane, which leads to its accumulation in the cytoplasm. Following this, β-catenin translocates to the nucleus and increases the transcription of Wnt-induced genes, including a Wnt inhibitor which promotes cardiac differentiation [2][5][6]. Taking advantage of the close association between these two pathways, a number of force aggregation techniques, such as hanging drop, microwell aggregation, and rotary suspension, have been employed to improve cardiac differentiation by altering cell adhesion dynamics [7][8][9][10].

Other mechanical factors which are known to influence cardiomyocyte differentiation include fluid shear stress, substrate rigidity, and cyclic stretch. Fluid shear stress, which is the frictional force exerted upon cells when fluid flows over them, is experienced very early on during development. Mouse embryonic stem cells (ESCs) subjected to long durations of fluid shear stress exhibit an increase in cardiac tissue specific markers, such as alpha-sarcomeric actin, smooth muscle actin, platelet-endothelial cell adhesion molecule-1, VEGF receptor 2, and smooth muscle protein 22-alpha [11]. A similar induction in the expression of cardiac markers, such as GATA-4 and alpha-MHC, was observed when embryonic stem cells were subjected to cyclic stretching (repetitive stretching and relaxation patterns that accompany cardiomyocyte contractility) [12]. As well as these biochemical changes, the typically elongated structure of cardiomyocytes is believed to be due to the effects of cyclic stretching [13].

The effects of substrate rigidity on the proliferation and differentiation potential of cells has been clearly established from a number of studies. The findings related to cardiomyocyte differentiation, however, have been contradictory; while one study noted enhanced cardiomyocyte differentiation from stem cells grown on a stiff matrix [14], another study pointed out that cardiac specification was driven by soft hydrogel substrates [15]. Working along similar lines, another research group that cultured embryonic stem cells on polyacrylamide hydrogel substrates demonstrated that substrate stiffness only influences the early specification of cardiac progenitor cells from the mesoderm and has no effect on the later stages, when the progenitor cells become irreversibly differentiated into cardiomyocytes. Moreover, their findings linked matrices of intermediate stiffness with maximal cardiomyocyte differentiation [16]. As well as influencing cardiomyocyte differentiation, it has also been shown that substrate stiffness can affect their functional properties. Cardiomyocytes cultured on hydrogels with a stiffness similar to that found in the body were produced action potentials with the longest duration compared to softer or harder substrates [17].

References

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