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
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.
SYNOPSIS: This is a project to develop cell-based therapies to treat cardiac arrythmias. 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 distinguished the cells from each other and working cardiomyocytes (CM). The specific code to be tested compares the fate of Nkx2.5 or Isl-1 expressing cells, using the early CCS marker HCN4 to distinguish CCS from CM. For example, cells co-expressing Nkx2.5 and HCN4 may derive the AV node, while those expressing Isl-1 and HCN4 may generate SA node tissue.
There are 3 aims:
1) Define the role of Nkx2.5 and Isl1 in development of the CCS. Previous work had shown that Nkx2.5 null embryos lack AV tissue, but this work was based on a single reporter line (mink: lacZ) for a gene that is a direct target of Nkx2.5, so it is not conclusive. The idea here is to cross HCN4:GFP mice into Nkx2.5 or Isl-1 null backgrounds. HCN4 encodes the If current and is the earliest known marker for conduction tissue. The PI will evaluate and compare the spatial arrangement, migration, growth, and number of HCN4:gfp cells, in addition to various IHC markers such as HCN1, mink, tbx3, connexion 40 and 45, in WT, heterozygous, and null backgrounds. In addition, the mutants will be crossed into the CCS-lacZ background (Glenn Fishman) which labels all the CCS.
2) Isolate Nkx2.5/HCN4 or Isl-1/HCN4 progenitor cells. This will be accomplished by first crossing nkx2.5/cre or isl-1/cre with a Z/Red reporter mouse and then to HCN4:gfp reporter mice to be able to track, isolate, FACS, and analyze the double-labeled cells. Electrophysiology will be carried out in addition to gene expression analysis.
3) Isolate the relevant human progenitors derived from hES and test function in a chick xenograft model. For this purpose hES reporter lines will be generated using the relevant Nkx2.5, isl-1, and HCN4 driven transgenes; the Laflamme et al. activin +bmp protocol will be used to generate CM in vitro; and these cells will then be explanted into chick embryos for integration and subsequent physiological analysis.
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. It is true that 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 arrythmias are important issues that this project seeks to impact. Significance is therefore very high. The clinical application is perhaps overstated. It is stated that congenital and acquired defects of cardiac pacemakers are causes of mortality in our society, and that therapeutically-delivered cells could be used to treat or prevent sudden cardiac death related to cardiac arrhythmias or congenital or acquired defects of cardiac pacemakers. Arrhythmias are due to ectopic or uncoordinated firing, and not generally due to the absence of SA, AV or HIS cells. It is also not established in the literature that replacement of these cell types by direct transplantation of viable SA, AV, or HIS cells in animal models in which these are destroyed results in functional improvements.
The proposal is generally feasible, and the applicant has done similar work laying the foundation for these studies during his cardiology fellowship with Ken Chien. In particular, their prior work identifies the expression pattern of the SA node which includes ISL1 and HCN4 expression. They also identify a dose-dependence of NKX2.5 AV for and HIS bundle development. Based on these studies, they hypothesize that a combinatorial action of transcription factors including NKX2.5 and ISL1 in conjunction with HCN4 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 Aim1 are straightforward and the PI should be able to accomplish a thorough description of the CCS phenotype for nkx2.5 and isl-1 null mice. Isl1 is preferentially expressed in the SA node, while Nkx2.5 is required in a dose-response manner for AV node tissue. The hypothesis fits well with current models for primary and secondary heart fields. The PI has also shown that loss of Nkx2.5 causes ectopic expression of HCN1 in the ventricle, and it is not entirely clear how these results fit with respect to the overall hypothesis. It will be important to look for stage-specific roles for these genes.
Experiments in Aim 2 are also reasonably straightforward and represent a very interesting fate-mapping approach. Dr. Wayne Giles is recruited for assistance in the electrophysiological analysis of purified progenitor populations.
Several minor issues were highlighted in Aims 1 and 2:
• One reviewer questioned how culture conditions will affect cellular phenotype.
• The applicant has yet to generate the GFP knockin HCN4 mice. Once these mice are generated, the remainder of the hypotheses to be tested in Aims 1 and 2 can be addressed.
• A potential difficulty may be encountered in isolating enough cells, which will result in a need for numerous embryos.
Heterogeneity in the SA and AV nodes as well as other parts of the conduction system are also acknowledged, and this heterogeity 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.
One of the mouse transgenic lines that is necessary for the proposed experiments in Aims 1 and 2, and the hESC reporter lines necessary for the experiments in Aim 3, are not yet available and need to be produced and validated
For Aim 2, the main concern is the massive breeding necessary to get 75-100 E8-9.5 triple heterozygotic transgenic embryos mice for the proposed studies.
Aim 3 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. First, the hES 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 field are not proposed. Moreover, although the culture conditions of Laflamme, et. al appear to generate a large number of cardiomyocytes, they may not be optimal conditions to generate a large number of cardiac conduction cell progenitors and therefore significant scaleup of hESC cultures may be necessary to generate enough cells for the chick embryo xenograft transplants. The best current protocol for generating cardiomyocytes yield 30% but these progenitors are likely to be only a small proportion of these. Results in this model are not likely to be useful for proof-of-principle for correcting arrhythmias since the embryonic environment is very different that a mature diseased heart. The Aim assumes conservation of mechanism from mouse to man, which seems reasonable.
Essential assistance comes from Dr. M. Pera and the USC hESC core facility. The applicant will presumably need help with the chick model and he should have arranged for this consultation. The SCRO needs at least to be notified, since WA09 hESCs are to be used in vitro and transplanted into chicken embryos; at present the SCRO form is all marked NO.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. Pashmforoush is well qualified to carry out the research. He is currently an Asst. Professor of Medicine at USC and affiliated with the Institute of Genetics and the Regenerative Medicine program. He is a physician scientist with training as a cardiac electrophysiology fellow. He was a productive postdoctoral fellow with Ken Chien at UCSD, where he published two first author papers in Nat Med (2001) and Cell (2004), the latter regarding the function of Nkx2.5 in cardiomyopathy. He has been an assistant professor of medicine at the University of Southern California for two years.
The applicant is well-trained in mouse models and has focused on molecular cardiac development over the past ten years; he thus has expertise to undertake the mouse transgenesis studies part of this project. He is in an excellent position to develop hES models as well. He currently receives independent extramural research support from the NIH to study the role of NKX2.5 in cardiac conduction system.
Although the investigator is considered quite strong to do this type of work, there is essentially no career development plan outlined. Although 80% of his time will be devoted to his research program, it is not clear to what extent Dr. Pashmforoush’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. He has the assistance of the USC ES core directed by Dr. Pera. A letter from Dr. Pera 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. Dr. Pera’s laboratory is located across the street from the Institute of Genetic Medicine, and the Stem Cell Institute’s resources are available for the project. Initially the hESC work will be done at the Stem Cell Institute. An additional letter of support is provided by Dr. Giles who will assist the investigator in performing electrophysiology studies on their isolated cell populations. The Chair (Dr. Crandall) and director of the Institute (Dr. Kedes) indicate enthusiasm and support for his work and 80% effort for research.
USC has a strong track record and commitment to stem cell biology and outstanding hES facilities and training potential, but it is not well defined in the institutional letter. The letter does highly support Dr. Pashmforoush and states that protected time and full institutional support are promised for 4 years from hire (Sept 2004), and that he had had a substantial start-up package. (His K08 award lasts until June 30,2009). 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, there was enthusiasm 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.