Reducing Heart Attack Scars in the Heart – Skip the Stem Cells

Two research groups have independently discovered that the heart scar that forms after a heart attack can be regenerated without stem cell treatments. Li Qian in the laboratory of Deepak Srivastava at the Gladstone Institute, and Victor Dzau’s team at Duke University have shown that the use of various molecules can trigger the conversion of scar tissue into heart muscle.

Dzau’s lab worked in mice and delivered microRNAs into fibroblasts, which are the cells that form the scar tissue in the heart. When these engineered fibroblasts took up the microRNAs, they became heart muscle cells.

MicroRNAs (miRNAs) are found inside cells are usually about 22 nucleotides long. These very small RNA molecules play important regulatory roles in animals and plants by targeting messenger RNAs (mRNAs) for cleavage or translational repression. Thus, miRNAs act as master regulatory molecules for gene expression (See Bartel DP. Cell. 2004;116(2):281-97).

“This is a significant finding with many therapeutic implications,” said Victor J. Dzau, MD, a senior author on the study who is James B. Duke professor of medicine and chancellor of health affairs at Duke University. “If you can do this in the heart, you can do it in the brain, the kidneys, and other tissues. This is a whole new way of regenerating tissue.”

After their experiments in tissue culture, Dzau’s lab showed that this conversion can also occur inside a living animal. Maria Mirotsou, PhD, assistant professor of cardiology at Duke and a senior author of the study commented, “This is one of the exciting things about our study. We were able to achieve this tissue conversion in the heart with these microRNAs, which may be more practical for direct delivery into cells and allow for possible development of therapies without using genetic methods or transplantation of stem cells.”

Since stem cells have proven difficult to manage inside the body, this mode of therapy has distinct advantages over stem cell-based treatments. Notably, the microRNA process eliminates technical problems such as genetic alterations, and also avoids the ethical dilemmas posed by the use of some stem cells.

“It’s an exciting stage for reprogramming science,” said Tilanthi M. Jayawardena, PhD, first author of the study. “It’s a very young field, and we’re all learning what it means to switch a cell’s fate. We believe we’ve uncovered a way for it to be done, and that it has a lot of potential.”

The next step is to test this approach in larger experimental animals. Dzau said therapies could be developed within a decade if additional studies advance in larger animals and humans.

“We have proven the concept,” Dzau said. “This is the very early stage, and we have only shown that is it doable in an animal model. Although that’s a very big step, we’re not there yet for humans.”

Gladstone researchers took a very different approach.  They delivered a cocktail of three genes that are known to direct cells to form heart muscle during embryonic development.  These three genes, Gata4, Mef2c and Tbx5, which are collectively called GMT, were placed into cells at the site of a heart attack.  Srivastava’s group engineered viruses to infect the heart tissue, and after inducing a heart attack, the engineered viruses were injected into the heart, at the site of the heart attack.

The heart contains several resident cell types that are not involved in contraction.  One of these resident populations is the fibroblast, which seems to be able to differentiate into heart muscle cells if properly coaxed.  The GMT-bearing viruses infected the resident fibroblasts and the infected cells differentiated into heart muscle cells that beat, formed connections with existing heart muscle cells, and contracted in synchrony.  The hearts that had suffered heart attacks came roaring back, functionally speaking, and were as good as new.

Dr. Qian, first author on this article, who is also a California Institute for Regenerative Medicine postdoctoral scholar and a Roddenberry Fellow. said, “These findings could have a significant impact on heart-failure patients—whose damaged hearts make it difficult for them to engage in normal activities like walking up a flight of stairs.  This research may result in a much-needed alternative to heart transplants—for which donors are extremely limited. And because we are reprogramming cells directly in the heart, we eliminate the need to surgically implant cells that were created in a petri dish.”

Dr. Srivastava noted, “Our next goal is to replicate these experiments and test their safety in larger mammals, such as pigs, before considering clinical trials in humans.  We hope that our research will lay the foundation for initiating cardiac repair soon after a heart attack—perhaps even when the patient arrives in the emergency room.”  Dr. Srivastava, is also a professor at the University of California, San Francisco (UCSF), with which Gladstone is affiliated.


Whitehead Scientists Discover Critical Role Played By One Enzyme In Embryonic Stem Cell Differentiation

Cells use gene expression programs to respond to external stimuli and maintain their present form and identity. Genes are stretches of DNA that encode a protein or RNA. Gene expression requires DNA sequences directly attached to the gene, and these sequences are called the “promoter” of the gene. The promoter is the binding site for the enzyme that executes the first step of gene expression. That enzyme is called “RNA polymerase.” RNA polymerase binds to the promoter and initiates the synthesis of an RNA copy of the gene, and process by which an RNA copy of the gene is synthesized rom the DNA template is called “Transcription.”

If the gene encodes a protein, the RNA is processed and sent from the nucleus of the cell to the cytoplasm, where protein/RNA complexes called “ribosomes” use the sequence in the RNA to make proteins that have amino acid sequences. Some genes, however, encode RNA molecules that are not used to direct the synthesis of protein, but are used for some other purpose.

Whatever the case, cells have a great deal of DNA in their nuclei. Almost every human cell, for example, contains so much DNA that if the DNA in one human cell was laid out end-to-end, it would stretch to a length of at least 1 meter. To pack all that DNA into the nucleus of a cell, the DNA is wound into a tight complex of DNA and proteins that is collectively called “chromatin.” Chromatin consists of DNA wound around proteins called histones, in ways that resemble the way thread is wound around a spool. These little histone spools are then wound into spirals that are then wound into a rosette of fibers. It is exceedingly for RNA polymerase to transcribe genes when they are wound into chromatin. How then are genes expressed? It turns out that particular proteins modify chromatin and cause it to loosen up so that RNA polymerase can access it.

Histone modifying proteins include those that encourage the formation of chromatin and tend to shut gene expression off (histone deacetylase, Polycomb-group proteins), and those that loosen chromatin and encourage gene expression (histone acetyl-transferases, histone methyltransferases). Therefore, we might expect to see such enzymes playing an important role in stem cell differentiation.

Therefore, we should not be surprised that stem cells researchers at the Whitehead Institute have discovered that a specific chromatin enzyme called lysine-specific demethylase 1 (LSD1) plays as embryonic stem cells differentiate into other cell types. Cell differentiation requires two key steps: 1) the genes active in the initial cell type must be deactivated; and 2) those genes important for the establishment of the new cell type must be activated. If the switch is not flawless, a transitioning cell may die or be driven to divide uncontrollably. Interestingly, LSD1 was known to be critical to development, but little was known about the key role it plays during differentiation, when cell-specific gene expression systems are switched on or off.

Paper author, Steve Bilodeau, who is also a postdoctoral research fellow in the laboratory of Whitehead Member, Richard Young, said; “We knew that cells express a new set of genes when the operating switch occurs. But this study shows it is also essential to shut off genes that were active in the prior cell state. If you don’t, the new cell is corrupted.”

Bilodeau and Warren Whyte, a Young lab graduate student and co-author, redefined LSD1’s role and described a previously unknown mechanism for silencing genes. They examined embryonic stem cell gene expression during differentiation and concentrated their efforts on those genes that must be shut off during differentiation. Whyte and Bilodeau found LSD1 was located on the promoters of those genes that had to be repressed in order for differentiation to occur. LSD1 was also found near DNA sequences called “enhancers,” which are associated with promoters and increase the ability of the promoters to activate gene expression.

What is LDS doing at the promoter and enhancer? When LSD1 receives the signal that the stem cell is going to differentiate, it transitions into an active conformation and silences those genes. Specifically, LSD1 hamstrings the ability of the enhancers of those genes to activate gene expression. With their enhancers rendered nonfunctional, transcription of these genes is silenced. While this occurs, other mechanisms switch on those genes necessary for the adoption of the new cell type.

Whyte added: “This reveals the critical function of LSD1 in cell differentiation. The enzyme decommissions the stem cell enhancers, thus allowing the new cell to function entirely within the parameters of the new operating system.”

Although this work focuses on one enzyme’s job in normal cells, Young sees broader implications, since LSD1 is a member of a class of molecules that regulate both gene activity and chromosome structure. Therefore, these findings about LSD1 could provide insights into how related regulators function. Similarly, understanding how a mechanism operates in normal cells provides a solid foundation for teasing apart what is going wrong in abnormal cells.

Young summed it up this way: This new knowledge brings us one important step closer to understanding defective operating systems in diseases such as cancer. And this may give us a new angle on drug development for these diseases.”

This work was published in “Enhancer decommissioning by LSD1 during embryonic stem cell differentiation;” Warren A. Whyte, Steve Bilodeau et al.; Nature, 2012; DOI: 10.1038/nature10805.