Heart disease is a leading cause of adult and childhood mortality. The underlying pathology is typically loss of heart muscle cells that leads to heart failure, or improper development of specialized cardiac muscle cells called cardiomyocytes during embryonic development that leads to congenital heart malformations. Because cardiomyocytes have little or no regenerative capacity after birth, current therapeutic approaches are limited for the over 5 million Americans who suffer from heart failure. Embryonic stem cells possess clear potential for regenerating heart tissue, but efficiency of cardiac differentiation, risk of tumor formation, and issues of cellular rejection must be overcome.
Our recent findings regarding direct reprogramming of a type of structural cell of the heart or skin called fibroblasts into cardiomyocyte-like cells using just three genes offer a potential alternative approach to achieving cardiac regeneration. The human heart is composed of muscle cells, blood vessel cells, and fibroblasts, with the fibroblasts comprising over 50% of all cardiac cells. The large population of cardiac fibroblasts that exists within the heart is a potential source of new heart muscle cells for regenerative therapy if it were possible to directly reprogram the resident fibroblasts into muscle cells. We simulated a heart attack in mice by blocking the coronary artery, and have been able to reprogram existing mouse cardiac fibroblasts after this simulated heart attack by delivering three genes into the heart. We found a significant reduction in scar size and an improvement in cardiac function that persists after injury. The reprogramming process starts quickly but is progressive over several weeks; however, how this actually occurs is unknown. Because this finding represents a new approach that could have clinical benefit, we propose to reveal the mechanism by which fibroblast cells become reprogrammed into heart muscle cells, which will be critical to refine the process for therapeutic use. We will do this by analyzing the changes in how the genome is interpreted and expressed at a genome-wide level at different time points during the process of fibroblast to muscle conversion, which represents the fundamental process that leads to reprogramming. The findings from this proposal will reveal approaches to refine and improve human cardiac reprogramming and will aid in translation of this technology for human cardiac regenerative purposes.
This research will benefit the state of California and its citizens by helping develop a new approach to cardiac regeneration that would have a lower risk of tumor formation and cellular rejection. In addition, the approach could remove some of the hurdles of cell-based therapy including delivery challenges and incorporation challenges. The mechanisms revealed by this research will enable refinement of the method that could potentially then be used to treat the hundreds of thousands of Californians with heart failure.
In this application, the Principal Investigator (PI) proposes to uncover the broad and progressive epigenetic changes that occur over time during the direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs). In the first aim, the PI will determine the genome-wide occupancy of key transcription factors at progressive stages of reprogramming. Next, in Aim 2, the PI will define the genome-wide epigenetic changes that occur during the cardiac reprogramming process. Finally, in the third aim, the PI will determine the transcriptional state of iCMs during progressive stages of reprogramming and will correlate this data with the transcription factor occupancy and epigenetic data collected in Aims 1&2.
Significance and Innovation:
- Reviewers viewed the proposal as a highly significant endeavor with potential benefits to the basic understanding of cell fate determination and its plasticity, as well as obvious clinical application.
- The proposal lacks concrete plans as to how the team will use this data to improve cardiac reprogramming efficiency or to achieve full differentiation to functional cardiomyocytes.
- There is innovation in that iCMs are novel. The proposed genome-wide studies are not particularly innovative but are appropriate for this stage of the research.
Feasibility and Experimental Design:
- Overall, the proposed experiments are feasible. The techniques employed are fairly standard molecular biological techniques and are well described.
- Alternate methods, should they become necessary, are adequately described.
-Reviewers expressed concern about the low efficiency of conversion to functional (beating) cardiomyocytes.
- The proposal is very clearly written with well-developed aims, approaches, methods, outcome analyses, and preliminary data.
- Preliminary data using human cells illustrate successful acquisition of cardiac marker expression in transcription factor-transduced fibroblasts but fall short of showing functional cardiomyocyte readouts.
- Reviewers expressed concern that preliminary data from mouse do not unequivocally establish the acquisition of a functional cardiomyocyte phenotype in the transcription factor-transduced cells and suggested that the proposed study is premature in the absence of further evidence of cell fate conversion.
- The facilities are perfectly appropriate to support these studies.
Principal Investigator (PI) and Research Team:
- The PI is an excellent scientist with an impeccable track record in innovative cardiovascular research.
- The research team is well suited to complete these studies, and the collaborating scientists are highly capable experts.
- Reviewers expressed some concern as to whether the PI will be able to commit the proposed 20% effort because of many other professional responsibilities.
Responsiveness to the RFA:
- Overall, the proposal addresses some aspects of the RFA, such as reprogramming. However, concerns exist regarding the proposed use of mouse cells. The potential impact of these studies on regenerative medicine will only become realized when the method is robust using human cells.
- A motion was made to move this application from Tier 1 into Tier 3, not recommended for funding. Some reviewers reiterated the concern that unequivocal evidence for conversion of fibroblasts to functional cardiomyocytes is still lacking and suggested that the proposed study is premature. However, the argument was made that the direct reprogramming of fibroblasts to cardiomyocytes represents important innovation in cardiovascular research. Taking into account the very strong track record of the PI, reviewers therefore felt the risk was worth taking. The motion was withdrawn.
- James Ellis
- Janet Rossant