Classical and novel pharmacological insights offered by the simple chick cardiomyocyte cell culture model: a valuable teaching aid and a primer for “real” research
https://goo.gl/uTiXYM
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).
https://goo.gl/uTiXYM
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).
β-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.
Furthermore, despite the apparent simplicity and robustness of this preparation, novel data can still be generated (7). Therefore, this technique is an ideal tool to give students an initial insight into the research process from constructing hypotheses, elaborating methods, and obtaining data to critically analyzing and potentially reporting their findings in peer-reviewed journals. This inclusion of undergraduate students as partners in collaborative research endeavors has been much discussed in the United Kingdom (UK) context (for a review, see Ref. 15) and is an ever-increasing feature of the student experience at UK Higher Education Institutions.
A typical trajectory for undergraduate students learning this technique in the authors’ own laboratories would first involve sessions on aseptic techniques and then the mechanics of enzymatically isolating viable populations of beating cardiac cells. Students usually observe the cell isolation procedure being conducted by their project supervisor on two or three separate occasions. They are told to write detailed notes of the procedure as it is being performed and then to come up with their own step-by-step guide to this method. Having produced their own guide, students undertake the cell isolation themselves with their project supervisor in close attendance offering verbal guidance and physical aid if necessary. This stage is repeated as often is necessary to convince the project supervisor that the students are competent enough to undertake the cell isolation procedure alone. In practice, this usually takes no longer than three supervised cell isolations. After this time, students are able to work unaided and independently of the project supervisor.
Once this has been successfully achieved and beating cells are routinely obtained, students are encouraged to design their own experiments around a suitable testable hypothesis. After data has been gathered, students analyze their data, subject it to statistical analysis, and present it graphically as plots and graphs. Finally, students are enjoined to discuss their own results with those already existing in the literature that might have relevance to the work they have completed. In the authors’ settings, these projects are finally presented by the students in the form of oral or poster presentations to an audience of peers and academic assessors.
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.
Furthermore, despite the apparent simplicity and robustness of this preparation, novel data can still be generated (7). Therefore, this technique is an ideal tool to give students an initial insight into the research process from constructing hypotheses, elaborating methods, and obtaining data to critically analyzing and potentially reporting their findings in peer-reviewed journals. This inclusion of undergraduate students as partners in collaborative research endeavors has been much discussed in the United Kingdom (UK) context (for a review, see Ref. 15) and is an ever-increasing feature of the student experience at UK Higher Education Institutions.
A typical trajectory for undergraduate students learning this technique in the authors’ own laboratories would first involve sessions on aseptic techniques and then the mechanics of enzymatically isolating viable populations of beating cardiac cells. Students usually observe the cell isolation procedure being conducted by their project supervisor on two or three separate occasions. They are told to write detailed notes of the procedure as it is being performed and then to come up with their own step-by-step guide to this method. Having produced their own guide, students undertake the cell isolation themselves with their project supervisor in close attendance offering verbal guidance and physical aid if necessary. This stage is repeated as often is necessary to convince the project supervisor that the students are competent enough to undertake the cell isolation procedure alone. In practice, this usually takes no longer than three supervised cell isolations. After this time, students are able to work unaided and independently of the project supervisor.
Once this has been successfully achieved and beating cells are routinely obtained, students are encouraged to design their own experiments around a suitable testable hypothesis. After data has been gathered, students analyze their data, subject it to statistical analysis, and present it graphically as plots and graphs. Finally, students are enjoined to discuss their own results with those already existing in the literature that might have relevance to the work they have completed. In the authors’ settings, these projects are finally presented by the students in the form of oral or poster presentations to an audience of peers and academic assessors.
General Teaching Points
The first experiments in the authors’ laboratories would involve adding the most potent sympathomimetic agonist, isoproterenol (ISO), to show the students the extent of the chronotropic changes this cell preparation can reveal. Addition of a nonselective β-adrenoceptor blocker, such as propranolol, would demonstrate the concept of competitive, reversible antagonism on the ISO response. These initial experiments are methodologically easy to undertake and conceptually simple for the students to understand. Indeed, such data are often displayed in graphical idealized terms in most introductory pharmacology textbooks. However, undertaking these experiments themselves gives students a real insight into fundamental concepts such as the efficacy and potency of drugs as well as a useful general introduction into primary cell culture techniques. Once mastery of these initial steps has been achieved, students can then progress to more complicated experiments either of their own design (in an ideal scenario) or with more guidance from their academic supervisor.
Under these circumstances, students have been able to obtain results that portray (extremely well) classical elements of receptor pharmacology such as full and partial agonism, competitive reversible antagonism, and inverse agonism. They have also been able to reveal some unusual aspects of β-adrenoceptor pharmacology by confirming the presence of a propranolol-insensitive low-affinity β1-adrenoceptor as well as a cardioinhibitory β3-adrenoceptor in these cells.
The first experiments in the authors’ laboratories would involve adding the most potent sympathomimetic agonist, isoproterenol (ISO), to show the students the extent of the chronotropic changes this cell preparation can reveal. Addition of a nonselective β-adrenoceptor blocker, such as propranolol, would demonstrate the concept of competitive, reversible antagonism on the ISO response. These initial experiments are methodologically easy to undertake and conceptually simple for the students to understand. Indeed, such data are often displayed in graphical idealized terms in most introductory pharmacology textbooks. However, undertaking these experiments themselves gives students a real insight into fundamental concepts such as the efficacy and potency of drugs as well as a useful general introduction into primary cell culture techniques. Once mastery of these initial steps has been achieved, students can then progress to more complicated experiments either of their own design (in an ideal scenario) or with more guidance from their academic supervisor.
Under these circumstances, students have been able to obtain results that portray (extremely well) classical elements of receptor pharmacology such as full and partial agonism, competitive reversible antagonism, and inverse agonism. They have also been able to reveal some unusual aspects of β-adrenoceptor pharmacology by confirming the presence of a propranolol-insensitive low-affinity β1-adrenoceptor as well as a cardioinhibitory β3-adrenoceptor in these cells.
Specific Teaching Points
A number of areas of uncertainty in the successful completion of any set of experiments have been evident in the many students who have undertaken this type of work. The first question invariably asked concerns the addition of cardioactive drugs. Should this be done by adding a small aliquot of a more concentrated stock solution to the existing media bathing the cells in the culture dish or should all the media be replaced by new media already containing the final concentration of the desired drug? The pros and cons of each approach can be debated, and students make the final choice largely depending on their analysis of the amount of data they can obtain by each approach. If adopting the former approach (as most students do), then apart from having to calculate volumes of drug that should be added to obtain appropriate final concentrations from an initial stock concentration, students also have to realize that the volume of drug added to the cells should not be so large as to significantly change the final volume of solution bathing the cells and thus dilute the final desired drug concentration.
Second, in terms of experimental design and efficient use of their time, students often have to think about the plating density of their freshly isolated cells when culturing them. This is because if the cells are plated too densely, the culture dishes will subsequently develop into a monolayer of cells all beating in unison. This restricts students to one experimental observation per culture dish. If the cells are plated too sparsely, few connections are formed between isolated groups of cells and little, if any, spontaneous beating activity may be observed. Previous experience has shown that using a cell plating density of 0.7–0.9 × 106 cells per 35-mm culture dish results in the formation of numerous, electrically separate groups of cells that beat independently of each other. This results in the number of observations per culture dish dramatically increasing. In the experiments outlined here, students usually obtained 6–8 million cells from 12 embryos and thus had at least 6 culture dishes full of separate groups of independently beating cells to experiment on per cell isolation cycle.
A number of areas of uncertainty in the successful completion of any set of experiments have been evident in the many students who have undertaken this type of work. The first question invariably asked concerns the addition of cardioactive drugs. Should this be done by adding a small aliquot of a more concentrated stock solution to the existing media bathing the cells in the culture dish or should all the media be replaced by new media already containing the final concentration of the desired drug? The pros and cons of each approach can be debated, and students make the final choice largely depending on their analysis of the amount of data they can obtain by each approach. If adopting the former approach (as most students do), then apart from having to calculate volumes of drug that should be added to obtain appropriate final concentrations from an initial stock concentration, students also have to realize that the volume of drug added to the cells should not be so large as to significantly change the final volume of solution bathing the cells and thus dilute the final desired drug concentration.
Second, in terms of experimental design and efficient use of their time, students often have to think about the plating density of their freshly isolated cells when culturing them. This is because if the cells are plated too densely, the culture dishes will subsequently develop into a monolayer of cells all beating in unison. This restricts students to one experimental observation per culture dish. If the cells are plated too sparsely, few connections are formed between isolated groups of cells and little, if any, spontaneous beating activity may be observed. Previous experience has shown that using a cell plating density of 0.7–0.9 × 106 cells per 35-mm culture dish results in the formation of numerous, electrically separate groups of cells that beat independently of each other. This results in the number of observations per culture dish dramatically increasing. In the experiments outlined here, students usually obtained 6–8 million cells from 12 embryos and thus had at least 6 culture dishes full of separate groups of independently beating cells to experiment on per cell isolation cycle.
