Classical and novel pharmacological insights offered by the simple chick cardiomyocyte cell culture model: a valuable teaching aid and a primer for “real” research
The chick embryo cardiomyocyte model of cell culture is a staple technique in many physiology and pharmacology laboratories. Despite the relative simplicity, robustness, and reproducibility inherent in this model, it can be used in a variety of ways to yield important new insights that help facilitate student understanding of underlying physiological and pharmacological concepts as well as, more generally, the scientific method. Using this model, this paper will show real data obtained by undergraduate students in the authors’ laboratories. It will first demonstrate classical pharmacological concepts such as full and partial agonism, inverse agonism, and competitive reversible antagonism and then move on to more complex pharmacology involving the characterization of novel receptors in these cells.
the chick embryo cardiomyocyte cell culture model has been a standard technique in teaching and research laboratories for a number of years across many countries. Such is its comparative straightforwardness that it may even be used as a teaching technique in high schools (16). The reason for its popularity is due to the relative ease with which students can obtain spontaneously beating sheets or separate colonies of electrically connected heart cells. This feature of the immature heart, the fact that isolated single cells will develop into beating groups of cells when cultured, lends itself greatly to studies of the different receptors that mediate changes in the beating rate of these cell cultures. Thus, chronotropic changes mediated via a variety of receptor populations can be easily studied (6, 9). Apart from these physiological and pharmacological observations, biochemical analyses can also be used to elucidate intracellular signaling pathways in these cells (11, 12).
The chick embryo cardiomyocyte model of cell culture is a staple technique in many physiology and pharmacology laboratories. Despite the relative simplicity, robustness, and reproducibility inherent in this model, it can be used in a variety of ways to yield important new insights that help facilitate student understanding of underlying physiological and pharmacological concepts as well as, more generally, the scientific method. Using this model, this paper will show real data obtained by undergraduate students in the authors’ laboratories. It will first demonstrate classical pharmacological concepts such as full and partial agonism, inverse agonism, and competitive reversible antagonism and then move on to more complex pharmacology involving the characterization of novel receptors in these cells.
the chick embryo cardiomyocyte cell culture model has been a standard technique in teaching and research laboratories for a number of years across many countries. Such is its comparative straightforwardness that it may even be used as a teaching technique in high schools (16). The reason for its popularity is due to the relative ease with which students can obtain spontaneously beating sheets or separate colonies of electrically connected heart cells. This feature of the immature heart, the fact that isolated single cells will develop into beating groups of cells when cultured, lends itself greatly to studies of the different receptors that mediate changes in the beating rate of these cell cultures. Thus, chronotropic changes mediated via a variety of receptor populations can be easily studied (6, 9). Apart from these physiological and pharmacological observations, biochemical analyses can also be used to elucidate intracellular signaling pathways in these cells (11, 12).
Adrenoceptor Signaling Pathways
In terms of the research interests of the authors, the work undertaken by their own students has focused on pharmacologically characterizing the contribution of four different β-adrenoceptor subtypes to cardiac contractility. The β-adrenoceptor family comprises members of the superfamily of protein receptors called G protein-coupled receptors (GPCRs). The Gs version of this protein links to a receptor (the β1-adrenoceptor and often the β2-adrenoceptor) that mediates stimulation of the activity of cardiac cells in terms of positive force generation, rate of contraction, and speed of relaxation (positive inotropy, chronotropy, and lusitropy, respectively). It does this largely through activation of a membrane-bound enzyme, adenylate cyclase, which produces increased amounts of an intracellular signaling molecule called cAMP. cAMP goes on to stimulate the production of more PKA, a molecule that can phosphorylate a number of cellular protein targets to change their function by this change in structure. Targets include the L-type Ca2+ channel (causing the entry of more Ca2+ into the cell, thus facilitating the positive inotropic and chronotropic changes) and phospholamban, a protein that regulates Ca2+ reuptake into the intracellular store for Ca2+, the sarcoplasmic reticulum (facilitating the positive lusitropic changes). There also exists a Gi version, which mediates negative inotropic and chronotropic effects (via the β3-adrenoceptor physiologically and the β2-adrenoceptor pathophysiologically in heart failure). In cardiac cells, this is done by the stimulation of a cytosolic enzyme, guanylate cyclase, which generates increased amounts of cGMP and consequently PKG. Phosphorylation events mediated by PKG include a reduction in the opening probability of the L-type Ca2+ channel (facilitating the negative inotropic and chronotropic changes).
Apart from the chronotropic analyses possible in spontaneously beating cardiac cell cultures, students have also undertaken biochemical studies looking at cAMP and PKA mobilization due to various β-adrenoceptor agonists. In addition, atomic absorbance analyses of media samples to look at Mg2+ efflux out of these cells in response to β-adrenoceptor stimulation has also been performed. Thus, it is evident that once students are routinely able to isolate spontaneously beating cells, there are a plethora of research avenues that could be pursued depending on their own interests and level of engagement.
In terms of the research interests of the authors, the work undertaken by their own students has focused on pharmacologically characterizing the contribution of four different β-adrenoceptor subtypes to cardiac contractility. The β-adrenoceptor family comprises members of the superfamily of protein receptors called G protein-coupled receptors (GPCRs). The Gs version of this protein links to a receptor (the β1-adrenoceptor and often the β2-adrenoceptor) that mediates stimulation of the activity of cardiac cells in terms of positive force generation, rate of contraction, and speed of relaxation (positive inotropy, chronotropy, and lusitropy, respectively). It does this largely through activation of a membrane-bound enzyme, adenylate cyclase, which produces increased amounts of an intracellular signaling molecule called cAMP. cAMP goes on to stimulate the production of more PKA, a molecule that can phosphorylate a number of cellular protein targets to change their function by this change in structure. Targets include the L-type Ca2+ channel (causing the entry of more Ca2+ into the cell, thus facilitating the positive inotropic and chronotropic changes) and phospholamban, a protein that regulates Ca2+ reuptake into the intracellular store for Ca2+, the sarcoplasmic reticulum (facilitating the positive lusitropic changes). There also exists a Gi version, which mediates negative inotropic and chronotropic effects (via the β3-adrenoceptor physiologically and the β2-adrenoceptor pathophysiologically in heart failure). In cardiac cells, this is done by the stimulation of a cytosolic enzyme, guanylate cyclase, which generates increased amounts of cGMP and consequently PKG. Phosphorylation events mediated by PKG include a reduction in the opening probability of the L-type Ca2+ channel (facilitating the negative inotropic and chronotropic changes).
Apart from the chronotropic analyses possible in spontaneously beating cardiac cell cultures, students have also undertaken biochemical studies looking at cAMP and PKA mobilization due to various β-adrenoceptor agonists. In addition, atomic absorbance analyses of media samples to look at Mg2+ efflux out of these cells in response to β-adrenoceptor stimulation has also been performed. Thus, it is evident that once students are routinely able to isolate spontaneously beating cells, there are a plethora of research avenues that could be pursued depending on their own interests and level of engagement.
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