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Exploiting natural mechanisms to derive human cardiomyocytes for modeling disease and regeneration

Funding Type: 
Basic Biology III
Grant Number: 
RB3-05171
Funds requested: 
$1 425 600
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Heart attacks are a major source of morbidity and mortality in humans. Unlike many organs and tissues of our bodies, human hearts do not regenerate. Instead, damaged heart muscle is replaced by scar tissue. Indeed, there is little evidence that cardiac stem cells or mature mammalian cardiac cells divide sufficiently to contribute to cardiac repair. In contrast, the hearts of salamanders and zebrafish exhibit remarkable potential to regenerate. We hypothesize that we can ‘take a page from the lowly newt (salamander)’ to learn to regenerate the human heart. Newts achieve this remarkable feat by a process of dedifferentiation. Non-dividing cells typical of mature tissues are induced to divide again by ‘lifting the brakes’ on cell division to make precise copies of themselves. The cells are pushed back just one step to a proliferative state while retaining their identity, or ‘sense of self’. Our strategy is to use a combination of novel molecular manipulations, already tested with success in our studies with another muscle, skeletal muscle of the limb. We will capitalize on our knowledge that newts completely lack a cell cycle ‘gatekeeper’ present in mammals and inactivate another during dedifferentiation. By mimicking this molecular mechanism and inactivating both cell cycle ‘gatekeepers’, we will induce differentiated nondividing cardiomyocytes to dedifferentiate, divide, and produce cells capable of repairing cardiac damage. One goal of this research is to grow cardiac cells in sufficient numbers to enhance our understanding of heritable human cardiac diseases and enable a characterization of the regulatory networks that have gone awry. These cells could also serve as material to screen for drugs that can overcome the genetically encoded malfunctions. As such, ‘dedifferentiation ala newts’ could potentially serve as an alternative to induced pluripotent stem cells (iPS), which are first reprogrammed to pluripotency and then induced to specialize again. To avoid uncontrolled growth and tumor formation, we will employ methods for ‘lifting the brakes’ only transiently, which we have successfully developed for skeletal muscle. Another goal of this research is to relieve the brakes on cell division in situ in the hearts of patients. The ability to cause healthy human cardiac cells to reproduce locally and replace damaged cardiac cells after a heart attack would constitute a novel therapy for heart disease. Finally, the lessons learned from the proposed novel ‘re-evolutionary’ approach to heart regeneration may prove broadly useful for other human tissues in addition to heart.
Statement of Benefit to California: 
Cardiovascular disease is the main cause of early death in the USA. According to the World Health Organization, the numbers will rise dramatically in the coming decades and have a profound impact on our social services and national health care system. Although advances in medical treatment have improved post-infarct survival, heart failure is an increasingly prevalent manifestation of cardiovascular disease. In California alone, the annual healthcare expenditures for stroke and heart attacks are staggering and exceed 700,000 cases, 3.5 million hospital days, and $34 billion (Source: American Heart Association). Clearly, an effective therapeutic approach that entails replacement of damaged myocardium with new cardiomyocytes is needed and would constitute a major advance. This grant proposal investigates a new regenerative approach which mimics a process that nature already successfully employs in other species. Newts are capable of regenerating their hearts, an ability that mammals have lost. We have elucidated an evolutionary difference between these species and defined a means by which the mechanism employed by newts can be achieved in the muscles of mammalian limbs. We propose here to employ this mechanism to regenerate the muscles of the human heart. The ability to grow cells from the human heart in culture, which is currently not possible, would also facilitate a deeper understanding of the etiology of heritable heart diseases and the discovery of novel therapeutic interventions. Furthermore, the ability to induce healthy cardiomyocytes in the vicinity of a heart attack to grow and replace the lost cardiomyoctyes could result in new functional heart tissue and prevent the development of obstructive scar tissue. If successful, this novel approach will advance the development of drugs for the treatment of heart disease and establish a means of cellular replacement of cardiac tissue in situ, leading to clinical therapies of enormous benefit for the citizens of California and beyond.
Review Summary: 
Project Synopsis: The goal of this proposed study is to investigate mechanisms by which fully differentiated mammalian cardiomyocytes (CM) could be induced to reenter the cell cycle and proliferate. This dedifferentiation will be achieved by interfering with cell cycle control genes or manipulating the cell’s microenvironment. In the first aim, the PI will explore the use of altered culture substrates to enhance the in vitro viability and proliferation of mouse and human CM from various sources. In Aim 2, the PI will determine the time course of cell cycle manipulation required for murine and human CM to re-enter the cell cycle and undergo cell division. The third aim will focus on isolation, expansion and characterization of single live cells to validate dedifferentiation of postmitotic CM. In Aim 4, the PI will model human genetic cardiac disease in vitro using dedifferentiation methods. The goal of the fifth aim is to test whether regeneration can be induced in vivo through manipulation of cell cycle control genes in a mouse model of myocardial infarction. Significance and Innovation: - The proposed studies are likely to provide fundamental biological insights. - Some reviewers thought that the proposal has potential for impacting regenerative medicine, since inducing CM to undergo even limited proliferation following cardiac damage could lead to new therapeutic options; other reviewers felt it is unclear if manipulation of cell cycle regulators, however transient, will ever be considered safe for therapeutic purposes. - The project is innovative, and the proposed strategy could be of importance to the wider stem cell community. Feasibility and Experimental Design: - Reviewers were concerned by the lack of preliminary data addressing the use of human CM and questioned the relevance and applicability of previous studies of skeletal myocytes. - Reviewers viewed the proposed experiments as technically feasible but noted that others had tried and failed to induce CM to re-enter the cell cycle. - Plans for use of dedifferentiated primary CM and strategies re-differentiation to the more mature state were not provided. - Reviewers were confused by an inconsistent designation of cell types throughout the proposal and unclear on the rationale for the use of CM derived from fibroblasts. - Reviewers found the study aims not well integrated with one another. Principal Investigator (PI) and Research Team: - The PI is an established leader in the field and a well-funded investigator with a very strong track record. - The research team has appropriate expertise; however, reviewers were concerned that there was no team member exclusively committed to the project Responsiveness to RFA: - The proposal is responsive to some features of the RFA, such as the use of human stem cells and analysis of molecular and cellular mechanisms. - The numerous experiments with murine cells are only marginally responsive to the objectives of the RFA.
Programmatic review: 
  • This application scored below the initial scientific merit funding line, no programmatic reason to fund the application was proposed, and the GWG voted to place the application in Tier 3, Not Recommended for Funding.
Conflicts: 

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