Heart disease is the leading cause of mortality and decline in the quality of life in the world. Current therapies are unable to restore function to damaged heart tissue. Recent scientific developments demonstrate the ability of human embryonic stem cells to form cardiomyocytes and this gives rise to the hope that we will be able to use these cells to differentiate and replace damaged myocardium. Despite this preliminary evidence and promise, we are far from being able to successfully accomplish regeneration of mature myocardium. This is largely due to our lack of understanding of all the factors that are involved in the differentiation process and our ability to manipulate the regeneration process. Our current understanding of the differentiation process indicates that there are at least four distinct stages of differentiation leading to cardiomyocytes, namely: (1) establishment of organizing centers; (2) mesendoderm induction; (3) establishment of cardiac precursors; and (4) terminal differentiation of beating cardiomyocytes. Our first quest is to identify what are the factors associated with each stage of differentiation leading to cardiomyocytes. We will carry out measurements of several biological macromolecules that are anticipated to play a role in sending signals for differentiation. This will be followed by systematic reconstruction of biochemical pathways so that we get a systems-level perspective on the differentiation process. In addition, we plan to embark on quantitative modeling of cardiomyogenesis to provide a firm basis for stem cell-based therapy. Quantitative modeling of cardiomyogenesis is certain to provide insights into the etiology of complex congenital heart defects (CHDs). A comprehensive quantitative approach will provide explanations for the malformations beyond that which can be obtained by single gene manipulations in typical experimental systems because of the ability to take into account the interconnected networks of signal transduction pathways. As important as this research is for pediatric cardiology, the significance for adult cardiology is perhaps more profound because it offers the promise of improving prospects for therapeutic intervention. In support of this view, adult cardiac disease, such as ventricular hypertrophy, reiterates molecular genetic pathways that are characteristic of embryonic development, and thus the predictions based on quantitative modeling and in vitro testing might define targets for pharmaceutical intervention.
Statement of Benefit to California:
In general, cardiovascular disease typically refers to a wide variety of heart and blood vessel diseases, including coronary heart disease, hypertension, stroke, and rheumatic heart disease. In 2000 nearly 200,000 Californians were hospitalized on account of heart disease. The statewide hospitalization rate due to heart disease in California is 6.4 per thousand in 2000. In addition to heart related illnesses, Stroke, hypercholesterolemia, high blood pressure and stroke accounted for another 30 percent of residents in California. Nearly 100,000 deaths resulted from these pathologies. In almost every case damage to the heart preceded death. Several risk factors besides genetics and heredity play a role in heart diseases. While prevention by healthy life styles and avoidance of risk factors is the first choice, this may not be feasible for all the residents of California. Ability of embryonic stem cells to differentiate into cardiac cells provides the hope that therapy by regeneration of damaged heart is a definitely possibility for the future. However, our understanding of the processes that lead a stem cell to differentiate into a cardiac lineage, is very primitive and any efforts at stem cell therapy mandate that we have a molecular and systemic understanding of cardiomyogenesis. This is the major objective of our proposal. Based on obtaining a detailed molecular picture of pathways that lead to cardiac differentiation from stem cells, we will be able to design small molecules/drugs that would trigger such differentiation in pathology. Further, such understanding will also aid preventive measures that will lead to reduction of the number of heart-related fatalities in California.
This project seeks to use a quantitative systems biology approach to define the signaling networks that control cardiomyocyte differentiation. The first goal is to generate a detailed signaling module map. Experiments in Aim 1 will attempt this by differentiating ESC in EB cultures using a comprehensive list of inhibitors of various signaling modules, followed by marker analysis at different times of EB development. The phospho-protein profiles will be determined by subcontract with a company. A rather long list of different cell lines will be used, starting with hES, mES, various mES reporter lines, and once generated a variety of hES reporter lines. SIGNIFICANCE AND INNOVATION Reviewer one: The project is innovative in the use of chemical biology and bioinformatics to develop testable models. There is in fact already quite a bit known about the signaling pathways (referred to as “legacy knowledge” and sample networks are already established, but this is a comprehensive view and could provide important information for key pathways. The concept derives from another project by the PI to predict cytokine release from a stimulated macrophage cell line. Reviewer two: This proposal to apply systems biology approaches to the cardiac myocyte differentiation process is very innovative and such an approach is likely necessary to fully understand that complex process. While many studies of cell differentiation require tremendous trial and error to define an optimal protocol, this study has the potential to make clear predictions of optimal methods with an underlying model accounting for the complex interactions. Successful completion of this work could rationalize the current hit-or-miss approach to lineage-specific differentiation – particularly for cardiomyocytes. To date, most analyses of cell differentiation have focused on gene regulation so a study that has a major emphasis on signaling pathways and protein phosphorylation is welcome. Futhermore, the dynamic modeling aspects are a novel addition. STRENGTHS: In principle the project has the potential to generate a comprehensive view of critical signaling pathways at different stages of ES-derived cardiomyocyte development. It brings together expertise from a variety of different disciplines. The proposal builds on the outstanding expertise of the PI in modeling complex biological processes. Furthermore, the inclusion of inhibitor approaches in the model-building provides causality information to complement the correlational information provided by the phosphoprotein analysis. The analysis of cell lines at different stages in the myocyte differentiation process is a further strength as is the inclusion of dynamic signaling components (i.e. time-dependent processes with lags) in the modeling. The collaboration with Dr. Mercola on analysis of the hESC differentiation is critical for the expertise both in ES cell handling and for generation of the required cell lines. WEAKNESSES: A major weakness is the lack of focus on a particular cell line. It seems unlikely that the experiments can be done on native hES because of the inefficient generation of CM, and the very significant heterogeneity of the EB system. The only way this project can succeed is with the use of defined reporter lines, so that the pathways specific to CM development can be traced. It does seem feasible to start this project using the currently available mES lines, eg. with Bra:GFP or Nkx2.5:GFP. The PI has submitted an NIH grant to do so, so this is clearly overlapping with the proposal. While the generation of similar hES lines is proposed, they will need to be validated. An important contributor to this project would Dr. Mercola and Belmonte, yet it is unfortunate that no letters of support are provided. Therefore, the only clear commitment from this strong group of investigators is 8% effort by Dr. Subramaniam, already PI on a large number of major projects. Overall, this proposal seems a bit premature. It is not clear how the balance of work on mouse vs human ES lines will be determined. As written, the bulk of the work could be on mESC which would not be responsive to the RFA. Given major differences between mouse and human cell lines, a proposal entirely focusing on hESC would be more compelling. Since many of the studies require sorting and isolation of specific cell populations, the proposal seems premature as the key GFP-expressing hESC lines are not yet available. Furthermore, the numbers of cells needed for the extensive analyses proposed is not discussed so feasibility is unclear. The prior work of the PI has depended on the experimental back-up of the Alliance for Cell Signaling. The ability of the PI’s team to perform the “wet-lab” experiments in this complex system is unproven. This is exemplified in part by the decision to “outsource” much of the phosphorylation analysis. The model outlined seems to be built in the format of a single cell. Given the complex cell-cell interactions that are critical for cardiomyocyte differentiation, it is surprising that the investigator didn’t develop a model explicitly including intercellular interactions. DISCUSSION: This is an innovative, but poorly developed proposal. The studies in human ESCs are premature - the development of human reporter line is believed to be a necessary first step for these experiments. These experiments should be done first in the better developed mouse system. In any case, the inefficiency of differentiation means that in both mouse and human systems, the investigator will be dealing with a heterogenous population in which it will be difficult to track network changes, unlike the homogeneous macrophage population with which the investigator has previously worked.