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RN1-00562-1: Transcriptional Regulation of Cardiac Pacemaker Cell Progenitors
Recommendation: Recommended for funding
Scientific Score: 72
First Year Funds Requested: $574,682
Total Funds Requested: $2,974,806
Public Abstract (provided by applicant)
Congenital and acquired defects of cardiac pacemakers are leading causes of morbidity and mortality in our society. Dysfunctions of the SA node and the lower conduction cells lead to a variety of complex arrhythmias that typically necessitate anti-arrhythmic therapy and implantation of devices. These treatments have significant limitations in their efficacy and risk-benefit ratio. Thus, it would be ideal to generate cell-based therapeutic approaches towards treating arrhythmias. Experimental data has provided compelling evidence that pacemaker and conduction cells of the heart separate early in development from the working myocardium and retain a relatively undifferentiated state. Prior cell-based approaches in regenerating myocardial damage in the heart have met limited success in part due to implantation of a diverse population of cells. This generally results in poor engraftment and undesirable outcomes. There is now evidence for resident conduction progenitor cells in myocardium that orchestrate the process of cell recruitment into the conduction tissue. In the current proposal we aim to identify the molecular events that lead to differentiation and formation of cardiac pacemaker cells. We will utilize the information obtained from the above experiments to generate cell based methods to treat cardiac arrhythmias. We aim to genetically manipulate the human embryonic stem cells so we can identify a selected population that is destined to become pacemaker cells. By replacing the cells responsible for normal beating of the heart, we hope to provide natural therapies for human conduction system disease
Statement of Benefit to California (provided by applicant)
The ultimate of goal of our proposal is identify a reliable mechanism for implementing a cell-based approach for treating human arrhythmias. Sudden cardiac death related to cardiac arrhythmia is a leading cause of morbidity and mortality in our society. The people of California have voted to implement new innovative ways of treating human disease by using human stem cells, the current project is in line with such wishes to create new therapeutic modalities towards treating heart disease.
Review
SYNOPSIS: This is a project to develop cell-based therapies to treat cardiac arrhythmias. It is focused on the isolation of progenitors for the cardiac conduction system (CCS) and specifically on cells that contribute to the AV or SA node tissue. The idea is that these cells are specified from a common cardiac progenitor (a hypothesis supported by earlier work from Mikawa and others) but that a transcriptional code distinguishes the cells from each other and working cardiomyocytes (CM). The specific transcriptional code to be tested compares the fate of two cardiac-specific subpopulations, using an early cardiac cell surface marker to distinguish CCS from CM.
The PI proposes to define the role of two cardiac-specific transcription factors in development of the CCS in mice and subsequently isolate CCS progenitor cells from such in mice.
The PI also proposes to generate human embryonic stem cells (hESCs), isolate the relevant human progenitors, and test function of these progenitors in a chick xenograft model.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The proposal is a series of complex but elegant studies focused mainly on mouse cardiac development, specifically on defining and isolating the progenitor cells that become the cardiac conduction system. The project relates to treating cardiac arrhythmia, which is a major contributor to heart failure and death. Most current approaches for cell-based therapies do not adequately consider the problem of cell diversity, the potential negative effect of too many or too few CCS cells in the explant, or the distinction of AV, SA fates. Heterogeneity of cell types, poor engraftment, and the risk of actually inducing arrhythmias are important issues that this project seeks to impact. Significance is therefore very high. The clinical application, however, is perhaps overstated, and may not be extendable to all types of cardiac arrhythmias.
The proposal is generally feasible, and the applicant has done similar work laying the foundation for these studies during his/her cardiology fellowship. Based on this prior work, the PI hypothesizes that a combinatorial action of transcription factors can identify a subpopulation of cardiac progenitor cells that are capable of forming cardiac pacemakers. The quality of the research plan is good, in particular aims 1 and 2 appear readily doable. The PI has an excellent track record in this field and has ample experience with mouse transgenic models.
The reviewers concur that this is a well-written and organized proposal clearly outlining experiments with precise endpoints, based on solid preliminary data and a strong logical hypothesis. The experiments in the first aim are straightforward and the PI should be able to accomplish a thorough description of the CCS phenotype for select cardiac-specific transcription factor(s) knockout(s) in mice. The hypothesis fits well with current models for primary and secondary heart fields. One reviewer noted that it will be important to look for stage-specific roles for these genes. Experiments in the second aim are also reasonably straightforward and represent a very interesting fate-mapping approach. A collaborator is recruited for assistance in the electrophysiological analysis of purified progenitor populations.
Several minor issues were highlighted:
- One reviewer questioned how culture conditions will affect cellular phenotype. - The applicant has yet to generate the mice that express a tag in conduction tissue. Once these mice are generated, the remainder of the hypotheses to be tested in the first and second aims can be addressed. - A potential difficulty may be encountered in isolating enough cells, which will result in a need for numerous embryos. - The hESC reporter lines necessary for the third aim are not yet available, and need to be produced and validated.
Heterogeneity in the SA and AV nodes as well as other parts of the conduction system is also acknowledged, and this heterogeneity may further complicate the ability to isolate an adequate number of cells. The in vitro studies to assess the growth and differentiation potential of these progenitors may require a large number of cells unless there is adequate scaledown of the culture methods.
For the second aim, the main concern is the massive breeding necessary to get 75-100 triple heterozygotic transgenic mice for the proposed studies.
The third aim clearly represents the more risky approaches and there is little if any preliminary data presented. However, it is a well-described and reasonable goal to develop a xenograft model, and there is precedence for it to work. The hESC reporter lines need to be generated, and this may require substantial tests of regulatory regions to get the best reporters. This may ultimately prove quite difficult, though the reporter lines would be valuable if produced. Methods to get around current roadblocks in this aspect are not proposed. Results in this model are not likely to be useful for proof-of-principle for correcting arrhythmias since the embryonic environment is very different from the environment in a mature diseased heart. The aim assumes conservation of mechanism from mouse to man, which seems reasonable.
Essential assistance comes from a collaborator and the institution’s hESC core facility. The applicant will presumably need help with the chick model and s/he should have arranged for this consultation.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The applicant is well qualified to carry out the research. The PI is currently an assistant professor of medicine, and is affiliated with the regenerative medicine program. The PI is a physician scientist with training as a cardiac electrophysiology fellow. S/he was a productive postdoctoral fellow, where s/he published two first author papers in two high-profile journals, the latter regarding the function of a specific transcription factor in a relevant cardiac disease. The applicant has been an assistant professor of medicine for two years.
The applicant is well-trained in mouse models and has focused on molecular cardiac development over the past ten years, and thus has expertise to undertake the mouse studies of this project. The applicant is in an excellent position to develop hESC models as well. S/he currently receives independent extramural research support from the NIH to study the role of a specific transcription factor in the cardiac conduction system.
Although the investigator is considered quite a strong candidate to do this type of work, there is essentially no career development plan outlined. Although 80% of the applicant’s time will be devoted to his/her research program, it is not clear to what extent the applicant’s development as an independent investigator will be devoted to stem cells and in particular hESC research. The PI seeks to develop translational approaches to treat heart disease with a focus on arrhythmias. A specific mentorship plan was not well-described and would be useful.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The PI is fully independent and has been provided with space for 8 researchers. The applicant has the assistance of the institution’s human embryonic stem cell core. A letter from the director of the hESC core, who is also a collaborator on the project, states that the institution will provide the investigator with quality-controlled stocks of hESC lines, and will help the PI in propagation and manipulation of these cells. The collaborator’s laboratory is located in close proximity, which will make resources available for the project. Initially the hESC work will be done at this collaborating institute. An additional letter of support is provided by second collaborator who will assist the investigator in performing electrophysiology studies on their isolated cell populations. The Chair and the Director of the collaborating institution indicate enthusiasm and support for his work.
The institution has a strong track record and commitment to stem cell biology and outstanding hESC facilities and training potential, but these are not well defined in the institutional letter. The letter does highly support the candidate and states that protected time and full institutional support are promised for 4 years from hire, along with a substantial start-up package. The applicant has a mentored career development award that lasts until 2009. This is a strength of the application. Many other investigators on campus have an interest in cardiac development, which overall establishes a fruitful environment for potential collaboration and cross-fertilization of ideas.
DISCUSSION: Among the reviewers, enthusiasm was strong for this highly significant project. The candidate is a well-published physician scientist with good electrophysiology training in a very good environment. Reviewers concurred that this was a very well-written proposal which is both innovative and feasible. Minor weaknesses were discussed, including: a lack of a mentorship plan; the need for large numbers of transgenic animals (75-100 was felt to be excessive and ambitious), and the fact that the very best current CM differentiation protocols yield only 30% cardiomyocytes, recognizing only a fraction of this population will be conduction sub-types needed for the study. Overall, these concerns did not dampen the enthusiasm of the innovative and significant proposal.
The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:
- None