Adult Directly Reprogrammed With Proteins into Cardiac Progenitor Cells Heal Heart After a Heart Attack and Make New Heart Muscle


Jianjun Wang from Wayne State School of Medicine in Detroit, Michigan and Xi-Yong Yu from Guangzhou Medical University and a host of graduate students and postdoctoral research fellows in their two laboratories have teamed up to make human cardiac progenitor cells (CPCs) from human skin fibroblasts through direct reprogramming. Direct reprogramming does not go through a pluripotent intermediate, and, therefore, produces cells that have a low chance of generating tumors.

To begin their study, Wang, and Yu and their colleagues isolated fibroblasts from the lower regions of the skin (dermis) and grew them in culture. Then they reprogrammed these cells in a relatively novel manner. This is a little complicated, but I will try to keep it simple.

Reprogramming cells usually requires scientists to infect cells with recombinant viruses that have been genetically engineered to express particular genes in cells or force cells to take up large foreign DNA. Both of these techniques can work relatively well in the laboratory, but you are left with cells that are filled with foreign DNA or recombinant viruses. It turns out that directly reprogramming cells only requires transient expression of specific genes, and once the cells have recommitted to a different cell fate, the expression of the genes used to get them there can be diminished.

To that end, some enterprising scientists have discovered that inducing cells to up modified proteins can also reprogram cells. Recently a new reagent called the QQ-reagent system can escort proteins across the cell membrane. The QQ-reagent has been patented and can sweep proteins into mammalian cells with high-efficiency and low toxicity (see Li Q, et al (2008) Methods Cell Biol 90:287–325).

Wang and Yu and their coworkers used genetically engineered bacteria to overexpress large quantities of four different proteins: Gata4, Hand2, Mef2c, and Tbx5. Then they mixed these proteins with their cultured human fibroblasts in the presence of the QQ reagent. This reagent drew the proteins into the cells and the fibroblasts were reprogrammed into cardiac progenitor cells (CPCs). Appropriate control experiments showed that cells that were treated with QQ reagent without these proteins were not reprogrammed. Wang and Yu and they research groups also exposed the cells to three growth factors, BMP4 and activin A, to drive the cells to become heart-specific cells, and basic fibroblast growth factor to turn the cells towards a progenitor cell fate.

The next set of experiment was intended to show that their newly reprogrammed were of a cardiac nature. First, the cells clearly expressed heart-specific genes. Flk-1 and Isl-1 are genes that earmark cardiac progenitor cells, and by the eighth day of induction, the vast majority of cells expressed both these genes.

 

Generation of protein-induced cardiac progenitor cells by modified transcript proteins. (A): Strategy of protein-induced cardiac progenitor cell (piCPC) generation. (B): Cell colonies were initially observed around days 4–8 and could be passaged to many small colonies around day 12. Representative phase contrast images are shown. The control was untreated human dermal fibroblasts in vehicle medium after 8 days. Scale bars = 100 μm. (C): quantitative polymerase chain reaction analysis of cardiac progenitor genes Flk-1 and Isl-1 in piCPCs. Fibroblast markers Col1a2 and FSP1 were also detected (∗, p < .05; ∗∗, p < .01 vs. day 0 control; error bars indicate SD; n = 3). (D): Representative fluorescent images are shown with typical cardiac progenitor markers Flk-1 (red) and Isl-1 (green) and fibroblast markers ColI (green) and FSP-1 (S100A4) (green) before and after reprogramming at day 8. DAPI staining was performed to visualize nuclei (blue) and all images were merged. Scale bars, 100 μm. (E): Flow cytometry analysis demonstrated Flk-1 and Isl-1 expressions were increased from d0 to d8 separately. Abbreviations: bFGF, basic fibroblast growth factor; BMP4, bone morphogenetic protein 4; ColI, collagen I; d, day; DAPI, 4′,6-diamidino-2-phenylindole; FSP1, fibroblast-specific protein 1; mGHMT, modified Gata4/Hand2/Mef2c/Tbx5.
Generation of protein-induced cardiac progenitor cells by modified transcript proteins. (A): Strategy of protein-induced cardiac progenitor cell (piCPC) generation. (B): Cell colonies were initially observed around days 4–8 and could be passaged to many small colonies around day 12. Representative phase contrast images are shown. The control was untreated human dermal fibroblasts in vehicle medium after 8 days. Scale bars = 100 μm. (C): quantitative polymerase chain reaction analysis of cardiac progenitor genes Flk-1 and Isl-1 in piCPCs. Fibroblast markers Col1a2 and FSP1 were also detected (∗, p < .05; ∗∗, p < .01 vs. day 0 control; error bars indicate SD; n = 3). (D): Representative fluorescent images are shown with typical cardiac progenitor markers Flk-1 (red) and Isl-1 (green) and fibroblast markers ColI (green) and FSP-1 (S100A4) (green) before and after reprogramming at day 8. DAPI staining was performed to visualize nuclei (blue) and all images were merged. Scale bars, 100 μm. (E): Flow cytometry analysis demonstrated Flk-1 and Isl-1 expressions were increased from d0 to d8 separately. Abbreviations: bFGF, basic fibroblast growth factor; BMP4, bone morphogenetic protein 4; ColI, collagen I; d, day; DAPI, 4′,6-diamidino-2-phenylindole; FSP1, fibroblast-specific protein 1; mGHMT, modified Gata4/Hand2/Mef2c/Tbx5.

Second, cardiac cells can differentiate into three different cell types: heart muscle cells, blood vessels cells, and smooth muscle cells that surround the blood vessels. In mesoderm progenitors made from embryonic stem cells, inhibition of the Wnt signaling pathway can drive such cells to become heart muscle cells (see Chen, et al Nat Chem Biol 5:100–107; Willems E, et al Circ Res 109:360–364; Hudson J, et al Stem Cells Dev 21:1513–1523). However, Wang, Yu and company showed that treating the cells with a small molecule called IWR-1 that inhibits Wnt signaling drove their cells to differentiate into, not only heart muscle cells, but also endothelial (blood vessel) cells and smooth muscle cells when the cells were grown on gelatin coated dishes. When left to differentiate in culture, the cells beat synchronously and released calcium in a wave-like fashion that spread from one cell to another, suggesting that some cells were acting as pacemakers and setting the beat.

 

Protein-induced cardiac progenitor cells (piCPCs) differentiated into three cardiac lineages: cardiomyocytes, endothelial cells, and smooth muscle cells. (A): Schematic representation of the strategy to differentiate piCPCs in differentiation medium with IWR1 factor. (B): Quantitative data of mRNA expression of cardiac lineage marker genes (∗, p < .05; ∗∗, p < .01; and ∗∗∗, p < .001 vs. day 0 control; error bars indicate SD; n = 3). (C): Immunofluorescent staining for MHC, MYL2, CD31, CD34, smMHC, and αSMA. The combination of the four factors, GHMT, induces abundant MHC and Myl2, and some expression of CD31 and smMHC 28 days after transduction. Nuclei were counter stained with DAPI. Scale bars = 100 μm. (D): Flow cytometry analysis for cTnI, CD31, and smMHC. mGHMT plus IWR1 significantly enhances cTnI expression, and, to a lesser extent, CD31 and smMHC expression. Abbreviations: αSMA, α-smooth muscle actin; BMP4, bone morphogenetic protein 4; cTnI, cardiac troponin I; cTnT, cardiac troponin T; d, day; DAPI, 4′,6-diamidino-2-phenylindole; GHMT, Gata4/Hand2/Mef2c/Tbx5; mGHMT, modified GHMT; MHC, myosin heavy chain; MYL2, myosin light chain 2; smMHC, smooth muscle myosin heavy chain.
Protein-induced cardiac progenitor cells (piCPCs) differentiated into three cardiac lineages: cardiomyocytes, endothelial cells, and smooth muscle cells. (A): Schematic representation of the strategy to differentiate piCPCs in differentiation medium with IWR1 factor. (B): Quantitative data of mRNA expression of cardiac lineage marker genes (∗, p < .05; ∗∗, p < .01; and ∗∗∗, p < .001 vs. day 0 control; error bars indicate SD; n = 3). (C): Immunofluorescent staining for MHC, MYL2, CD31, CD34, smMHC, and αSMA. The combination of the four factors, GHMT, induces abundant MHC and Myl2, and some expression of CD31 and smMHC 28 days after transduction. Nuclei were counter stained with DAPI. Scale bars = 100 μm. (D): Flow cytometry analysis for cTnI, CD31, and smMHC. mGHMT plus IWR1 significantly enhances cTnI expression, and, to a lesser extent, CD31 and smMHC expression. Abbreviations: αSMA, α-smooth muscle actin; BMP4, bone morphogenetic protein 4; cTnI, cardiac troponin I; cTnT, cardiac troponin T; d, day; DAPI, 4′,6-diamidino-2-phenylindole; GHMT, Gata4/Hand2/Mef2c/Tbx5; mGHMT, modified GHMT; MHC, myosin heavy chain; MYL2, myosin light chain 2; smMHC, smooth muscle myosin heavy chain.

Then these cells were transplanted into the heart of mice that had suffered heart attacks. When compared to control hearts that received fluid, but no cells, the hearts of the animals that received protein-induced CPCs showed decreased scarring by 4 weeks after the transplantations. They also showed the growth of new heart muscle. A variety of staining experiments established that the engrafted protein-induced CPCs positive for heart muscle- and endothelial-specific cell markers. These experiments showed that transplantation of cardiac progenitor cells can not only help attenuate remodeling of the left ventricular after a heart attack, but that the protein-induced CPCs (piCPCs) can develop into cells of the cardiac lineage.

In vivo delivery of protein-induced cardiac progenitor cells improves cardiac function after myocardial infarction. (A): EF, FS, LVDd, and LVDs were analyzed by echocardiography (∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001 vs. relevant 1 week; all data are presented as mean ± SD, n = 8). (B): Transplanted cells were detected by magnetic resonance imaging 4 weeks after myocardial infarction (MI). Red arrow points to the signal loss due to SPIO-labeled cells. (C): Masson trichrome staining on heart sections 4 weeks after MI injection in sham, vehicle, and piCPC groups. Scale bar = 0.5 cm. (D): Immunofluorescent staining for cTnI (red), CD31 (red), and anti-dextran (SPIO, green) of heart sections after piCPCs were transplanted 4 weeks after MI. White arrows point to transplanted cells or colocalization of cTnI or CD31 with SPIO. Scale bars = 100 μm. Abbreviations: cTnI, cardiac troponin I; DAPI, 4′,6-diamidino-2-phenylindole; EF, ejection fraction; FS, fractional shortening; LVDd, left ventricular internal diameter at end-diastole; LVDs, left ventricular internal diameter at end-systole; piCPCs, protein-induced cardiac progenitor cell; SPIO, superparamagnetic iron oxide; W, week.
In vivo delivery of protein-induced cardiac progenitor cells improves cardiac function after myocardial infarction. (A): EF, FS, LVDd, and LVDs were analyzed by echocardiography (∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001 vs. relevant 1 week; all data are presented as mean ± SD, n = 8). (B): Transplanted cells were detected by magnetic resonance imaging 4 weeks after myocardial infarction (MI). Red arrow points to the signal loss due to SPIO-labeled cells. (C): Masson trichrome staining on heart sections 4 weeks after MI injection in sham, vehicle, and piCPC groups. Scale bar = 0.5 cm. (D): Immunofluorescent staining for cTnI (red), CD31 (red), and anti-dextran (SPIO, green) of heart sections after piCPCs were transplanted 4 weeks after MI. White arrows point to transplanted cells or colocalization of cTnI or CD31 with SPIO. Scale bars = 100 μm. Abbreviations: cTnI, cardiac troponin I; DAPI, 4′,6-diamidino-2-phenylindole; EF, ejection fraction; FS, fractional shortening; LVDd, left ventricular internal diameter at end-diastole; LVDs, left ventricular internal diameter at end-systole; piCPCs, protein-induced cardiac progenitor cell; SPIO, superparamagnetic iron oxide; W, week.

These are exciting results. It shows that direct reprogramming can occur without introducing genes into cells by means that can complicate the safety of the implanted cells. Also, because the cells are differentiated into progenitor cells, they still have the ability to proliferate and expand their numbers, which is essential for proper regeneration of a damaged tissue.

After a heart attack, the ventricle wall scars over and can become thin. However, piCPCs that have been directly reprogrammed from mature, adult cells can be used to replace dead heart muscle in a living animal.

Despite these exciting advances, further questions remain. For example, are the physiological properties of cells made from piCPCs similar enough to match the functional parameters of the heart into which they are inserting themselves? More work is necessary to answer that question. Functional equivalence is important, since a heart that does not function similarly from one end to the other can become arrhythmic, which is clinically dangerous. Further work is also required to precisely determine how well cells derived from piCPCs mature and coupling with neighboring cells. Therefore, larger animal studies and further studies in culture dishes will be necessary before this technique can come to the clinic. Nevertheless, this is a tremendous start to what will hopefully be a powerful and fruitful technique for healing damaged hearts.

Doubts About Cardiac Stem Cells


Within the heart resides a cell population called “c-kit cells,” which have the ability to proliferate when the heart is damaged. Several experiments and clinical trials from several labs have provided some evidence that these cells are the resident stem cell population in the heart that can repair the heart after an episode of cardiac injury.

Unfortunately, a few new studies, and in particular, one that was recently published in the journal Nature, seem to cast doubt on these results. Jeff Molkentin of Cincinnati Children’s Hospital Medical Center and his co-workers have used rather precise cell lineage tracing studies in mice to follow c-kit cells and their behavior after a heart attack. His results strongly suggest that c-kit cells rarely produce heart muscle cells, but they do readily differentiate into cardiac endothelium, which lines blood vessels.

“The conclusion I am led to from this is that the c-kit cell is not a cardiac stem cell, at least in term of its normal, in vivo role,” said Charles Murry, a heart regeneration researcher at the University of Washington who was not involved in this study.

Molkentin’s study is what some stem cells researchers are calling the nail in the coffin for c-kit cells. In fact the Molkentin paper is simply the latest in a series of papers that were unable to reproduce the results of others when it comes to c-kit cells. Worse still, one of the leading laboratories in the c-kit work, Piero Anversa at Harvard Medical School, has had to retract on of this papers and there is also some concern about his publication regarding the SCIPIO trial. Eduardo Marbán, an author of the new study and a cardiologist at the Cedars-Sinai Heart Institute in Los Angeles, said, “There’s been a tidal wave in the last few weeks of rising skepticism,” Nevertheless, the present dispute is not yet settled, and many scientists still regard the regenerative powers of c-kit cells as a firmly established fact.

In his laboratory, Piero Anversa and his colleagues and collaborators have shown that c-kit cells—cardiac progenitor cells expressing the cell surface protein c-kit—can produce new heart muscle cells (cardiomyocytes). Anversa and others also helped usher these cells, which are also known as CPCs or cardiac progenitor cells, into clinical trials to test whether they might help repair damaged cardiac tissue. This culminated in the SCIPIO trial, which showed that patients treated with their own CPCs showed long-lasting and remarkable improves in heart function.

Follow-up work by other research teams, however, has not been able to confirm these studies, and their work has raised doubts about the potential of c-kit cells to actually build new heart muscle. In his contribution to the c-kit controversy, Molkentin and his colleagues genetically engineered mouse strains in which any c-kit-expressing cells and their progeny would glow green. To do this, they inserted a green fluorescent protein gene next to the c-Kit locus. Therefore any c-kit-expressing cells in the heart would not only glow green, but whatever cell type they differentiated into would also glow green. After inducing heart attacks in these mice, Molkentin and others discovered that only 0.027 percent of the heart muscle cells in the mouse heart originated from c-kit cells. “C-kit cells in the heart don’t like to make myocytes,” Molkentin told The Scientist. “We’re not saying anything that’s different” from groups that have not had success with c-kit cells in the past,” Molkentin continued, “we’re just saying we did it in a way that’s unequivocal.”

Molkentin’s study did not address why there’s a discrepancy between his results and those of Anversa and another leader in the c-kit field, Bernardo Nadal-Ginard, an honorary professor at King’s College London. Last year in a paper published in the journal Cell, Nadal-Ginard and his colleagues showed that heart regeneration in rodents relies on c-kit positive cells and that depleting these cells abolishes the regenerative capacity of the heart.

In an email to a popular science news publication known as The Scientist, Nadal-Ginard suggested that technical issues with Molkentin’s mouse model could have affected his results, causing too few c-kit cells to be labeled. Additionally, “the work presented by Molkentin used none of our experimental approaches; therefore, it is not possible to compare the results,” Nadal-Ginard said in an e-mail.

Anversa said his lab is working with the same mouse model Molkentin used, “but our data are too preliminary to make any specific comment. Time will tell.”

Molkentin’s paper seems point to further problems with Aversa’s work with c-kit cells.  Last month, one of the papers Anvera and his group had published in the journal Circulation had to be retracted because the data used the write that paper were “sufficiently compromised.”  Then a few days later, the paper describing the results of the SCIPIO study that appeared the journal The Lancet expressed concern about supplemental data that was included with the published results.  These data came from the human clinical trial that treated heart patients with their own c-kit cells.  Harvard Medical School and Brigham and Women’s Hospital are investigating what went wrong with this study and the publication itself.

Regardless of Anversa’s present tribulations, Marbán is advancing another type of heart-specific stem cell, called cardiosphere-derived cells or CDCs.  Marbán and his colleagues have already used CDCs in a human clinical trial known as the CADUCEUS trial.  In this trial, heart attack patients treated with CDCs saw their heart scars shrink.  Marbán said he had been a true believer in c-kit cells, until the data started mounting against them. “The totality of the evidence now says the c-kit cell is no longer a cardiomyocyte progenitor,” he said.

Now even if c-kit cells do not make new heart muscle, it is possible that they heal the heart through other means.  The patients in the SCIPIO trial saw real, genuine improvements in their heart function and these results cannot be so cavalierly dismissed.  In fact, Murry said that just because the mechanistic basis for the human study remains in doubt, promising clinical results should not be dismissed. “Those results can be considered independent,” he said.  Molkentin also added that it’s possible that c-kit cells work in unknown ways to repair heart tissue.  Since clinical treatments involves high levels of c-kit cells that have been immersed in culture conditions, “Perhaps these cells act a little different,” Molkentin said.

Nadal-Ginard also noted that discrepancies do exist between his data and those of others, and that these discrepancies should not be papered over, but should be robustly debated and addressed.  He said he’d be willing to work with Molkentin to get to the bottom of it. “The concept under dispute is too important for the field of regenerative medicine—and regenerative cardiology, in particular—to turn into a philosophical/dogmatic argument instead of settling it in a proper scientific manner.”  Here here.

A Powerful Tool For Repairing Damaged Hearts


A new report from Johns Hopkins University researchers indicates that a particular stem cells that helps build mouse hearts can self-renew. This discovery, which might very well apply to humans as well, could potentially open inroads to use these cells to repair hearts damaged by disease, or, perhaps, even grow new heart tissue for transplantation.

This study is slated for publication in the journal eLife. Chulan Kwon, Ph.D., an assistant professor of cardiology and member of the Institute for Cell Engineering at the Johns Hopkins University School of Medicine and his team, found that during heart formation, these so-called cardiac progenitor cells or CPCs proliferate, but do not differentiate into heart cells in an embryonic structure known as the second pharyngeal arch. This insight into the biology of CPCs may contribute to better understanding of how to prevent and treat congenital heart defects.

Kwon noted that, “Our finding that CPCs are self-renewing—that they can keep dividing to form new CPCs—means they might eventually be maintained in a dish and used to make specific types of heart cells.”

Kwon continued: “Growing such cells in a dish would be an enormous step toward better treatment for heart disease.”

Kwon’s laboratory initially tackled the elucidating the contribution of two genes, Numb and Numbl, in the CPC biology. Other studies have shown that these two genes are required to guide stem and progenitor cells to their fully mature, specialized functions. Numb and Numbl are highly conserved in mice and humans (i.e. the proteins encoded by these genes in mice and humans are nearly identical). This conservation indicates that Numb and Numbl are probably doing something very important the lives of CPCs.

As a first step, Kwon and others made loss-function mutations in Numb and Numbl. The results were striking. According to Kwon, embryos that lacked functional Numb and Numbl protein, “failed to develop normal hearts and died at an early stage of development, showing us that Numb and Numbl are needed for CPCs to build the heart.”

With the crucial role of Numb and Numbl in the lives of CPCs in mind, Kwon and his colleagues tried to determine the location of CPCs in the developing embryo. For these experiments, they used mouse embryonic stem cells that lacked functional Numb and Numbl, and expressed a glowing red protein in all CPC cells. Such a glowing red protein would instantly give away the CPCs’ location. These embryonic stem cells have the ability to integrate into a growing embryo, but the absence of functional Numb and Numble proteins in these cells prevents them from growing into a viable embryo.

Next, Kwon’s group injected these engineered embryonic stem cells into viable mouse embryos at the blastocysts stage. The blastocyst stage forms early during mammalian development, and it consists of the two cell populations that will form the embryo (inner cell mass cells) and the placenta (trophoblast cells). “The normal cells in these blastocysts compensated for those that lacked Numb and Numbl, allowing the resulting embryos to survive,” Kwon says.

Once these chimeric embryos began to grow, Kwon’s group examined them for red-glowing cells. They found the glowing red cells in the second pharyngeal arch, which is known for forming parts of the neck and face. Kwon says their study is the first to identify the second pharyngeal arch as the home of the CPCs.

pharyngeal_arches

 

The cells of the second pharyngeal arch go on to form the stapes in the middle ear and the stapedius muscle that attaches to the stapes.

Pharyngeal_arch_cartilages

Additionally, Kwon’s group cultured second pharyngeal arch cells with CPCs. They discovered that the cultured CPCs self-renewed without developing into specialized heart cells. This is potentially an important step toward using CPCs to treat heart disease.

The next step, he says, is to direct the laboratory-grown CPCs to form new heart tissue that could be used to regenerate disease-damaged heart tissue. “Eventually, we might even be able to deliver cells to damaged hearts to repair heart disease,” Kwon says.

Beta Blockers and Cardiac Progenitor Cells


The heart receives nerve input from several nerves. Some of these inputs come from the branches of the autonomic nervous system. If that sounds cryptic, just think of the word “automatic.” In other words, the things your body does without you consciously thinking about it are largely directed by the autonomic nervous system: digestion, breathing, the beating of your heart, and so on are all things that our body does without us consciously thinking about it.

The autonomic nervous system consists of two branches, the sympathetic and the parasympathetic branches of the autonomic nervous system. With respect to the heart, the sympathetic nerve inputs to the heart accelerate the heart beat and the force of the heart’s contractions. The parasympathetic inputs to the heart slow the heartbeat, but do not have any direct effect on the force of the heart’s contractions.

autonomic innervation of the heart

The sympathetic nerves that connect to the heart release the neurotransmitters epinephrine and norepinephrine. These neurotransmitters bind to receptors on the surface of heart muscle cells in order to elicit their stimulatory responses. The receptors that bind epinephrine and norepinephrine are called “adrenergic” receptors because they bind epinephrine, which used to be called “adrenaline.” When pharmacists talk about “adrenergic” stimulation, they mean receptors that bind to epinephrine and norepinephrine (for the sake of brevity, I am going to abbreviate these two molecules as Epi/NE).

Activation of Beta2 resize bronchial tubes

Now if all this seems confusing, I am sorry, but it is going to get worse. You see there are different flavors of adrenergic receptors. There are alpha and beta adrenergic receptors. Both alpha and beta adrenergic receptors bind Ep/NE, but the specific responses they elicit can differ, depending on the cell and the machinery it has to respond to the bound receptor. A quick example might help make this clear. If you get an asthma attack, you can breathe in a product called Primatene Mist, which is simply aerosolized epinephrine. Epi, in your lungs, causes the smooth muscles that surround your breathing passages to relax and your breathing passages dilate. This allows you to breath much more easily. However, that same molecule, Epi, will cause your heart to beat faster and harder. The same molecule – Epi – elicits two completely distinct responses from two tissues. This is due to the fact that the heart has one type of adrenergic receptor on the surfaces of its cells (so-called beta1 adrenergic receptors), and the bronchial smooth muscle has a distinct beta adrenergic receptor the on the surfaces of its cells (so-called beta2 adrenergic receptors).

I realize that this is a very long introduction, but it is necessary in order to talk about the paper that I found. In this paper, scientists in Mark Sussman’s laboratory at the San Diego Heart Research Institute have examined cardiac progenitor cells (CPCs) from male mice and their response to beta adrenergic stimulation. You see, once we are born, adrenergic stimulation causes the heart to grow and mature. However, once the heart muscle cells mature, this stimulation no longer causes the heart to enlarge in the same way that heart normally does shortly after birth, although the heart is still capable of remodeling in response to constant aerobic exercise. However, after a heart attack, the secretion of Epi/Ne tends to drive deterioration of the heart. Therefore, a common drug strategy to treat heart attack patients is to prescribe a class of drugs called “beta blockers,” which protect the heart from the deleterious effects of adrenergic stimulation after a heart attack. However, the effects of adrenergic stimulation on CPCs is unknown, and Sussman’s laboratory used cultured CPCs to determine the effects of adrenergic stimulation on CPCs.

CPCs are a stem cell population that resides in the heart. A respectable corpus of literature has shown that CPCs can differentiate into various heart-specific cell types and replace dying heart muscle. Our hearts do not recover properly after a heart attack because the CPCs healing capacities are overwhelmed after a heart attack (See Leri A, Kajstura J, and Anversa P, Circulation Research 109 (2011) 941-61 for an excellent summary of the physiological tasks performed by CPCs).

In the Sussman paper, cultured CPCs from mice and humans were cultured in the laboratory.  It was quickly discovered that CPCs do NOT express beta1 adrenergic receptors on their surfaces, but beta2 adrenergic receptors.  You might smirk and this and say “so what?”  However this is significant for the following reason:  Early in their lives, heart muscle cells expression beta2 adrenergic receptors, but they later switch to exclusive expression of beta1 adrenergic receptors.  They express beta2 adrenergic receptors during that time when they can rapidly divide and respond to the needs of the heart.  CPCs express beta 2 adrenergic receptors only when they are in their undifferentiated state.  Once they differentiate, they switch to beta1 adrenergic receptors.

Secondly, Sussman and his crew discovered that stimulation of the beta2 adrenergic receptors on the surfaces of CPCs caused them to divide.  Sussman and others used a molecule called fenoterol, which binds very tightly to beta2 adrenergic receptors and activates them.

Third, once the CPCs were differentiated into heart muscle cells, they no longer expressed beta2 adrenergic receptors, but expressed beta 1 adrenergic receptors.  Did this change the response of the cells to adrenergic stimulation?  YES.  Instead of dividing in response to adrenergic stimulation, the cells were much more sensitive to dying.  To make sure that this result was not a fluke, Sussman and others engineered CPCs to express beta1 adrenergic receptors, and, sure enough, those cells were also sensitized to cell death upon expression of beta1 adrenergic receptor.

This is all fine and dandy for a culture dish, but can this make a difference in a living animal?  Sussman used a specific mouse strain called TOT.  These mice have a special pathology in that their hearts enlarge and start to not work very well once they are exposed to large quantities of Epi/NE.  Can beta blockers prevent this enlargement of the heart in TOT mice?  It definitely can.  However, Sussman wanted to know what happened to the CPCs.  Therefore, they broke the mice into three groups.  Two groups received metoprolol and the third did not.  Then four weeks later, one TOT mouse group that had received metoprolol and another that had not received transplantations of marked CPCs into their hearts (the CPCs glowed).  Then they examined the CPCs two weeks after the implantation.  The CPCs in non-metoprolol-treated TOT mice took a beating.  However, in the metoprolol-treated mice, the CPCs were three times more prevalent and showed overall lower levels of programmed cell death.  There was less DNA synthesis in the hearts of metoprolol-treated animals, indicating that there was less of a need for replacement of dead cells.

These results indicate that beta blockers do more than protect the heart from excessive Epi/NE after a heart attack.  They also protect the CPCs in the heart, and that could be an even more significant contribution to the life of the heart after a heart attack.  It is might be possible to direct or even augment the activity of CPCs in the heart after a heart attack to accelerate cardiac healing.  That would be a tremendous step in cardiac healing.

Growth Factors to Heal the Heart


When the heart suffers a heart attack, local areas of the heart experience cell death as a result of blockage in a coronary vessel. The cell death is followed by local inflammation which causes further cell death and produces a heart scar. This produces a situation in which a portion of the heart does not contract and also does not conduct impulses to beat. Can this dead heart tissue live again?

Several experiments have used stem cells to refurbish the dead heart tissue, and a variety of different stem cells can clearly produce new heart cells that help the heart beat better. Can growth factors that stimulate cell growth and division do a similar job?

Just injecting growth factors into the bloodstream will not do because the growth factors will not spend any appreciable time in or around the heart cells. Is there another way to do it? Yes. The answer is hydrogels.

Hydrogels are semi-solid materials that can be made and in which the growth factors can be embedded. The hydrogels are gradually degraded while they release growth factors into the heart tissue. The slow but stead release of various growth factors can induce the heart to heal itself.

Works from the laboratory of Michael E. Davis at Georgia Institute of Technology and Emory University School of Medicine in Atlanta, Georgia have published a paper in PLoS ONE describing this very strategy. Using rats that had suffered heart attacks, Davis and his group applied a polyethylene glycol-based hydrogel laced with two growth factors, hepatic growth factor (HGF) and vascular endothelial growth factor (VEGF) to the hearts of these animals.

There were no immediate effects to the application of these hydrogels as determined by electrocardiograms. However, with the passage of time, some remarkable changes to the hearts of these rats were observed. Three weeks after the application of hydrogels to rat hearts, animals treated hydrogel material only, injected with growth factors only showed no significant improvement over those rats that were not injected with anything. But those rats whose hearts had been injected with hydrogels laced with VEGF showed a 50% increase in blood vessel density and those injected with hydrogel imbued with HGF and VEGF showed a 100% increase in blood vessel density. These same rats also showed a huge reduction in the size of the heart scar (41.5 % vs 13.9% fibrosis), and also showed significant increased in heart function after three weeks.

Why did these growth factors work so well? Several experiments conducted by Davis’ group showed that the stem cell population in the heart, the cardiac progenitor cells or CPCs, were pitched into overdrive by the growth factors, In short, in the presence of these two growth factors, the cells went nuts. They went to area where the hydrogel had been applied and made new heart muscle cells and blood vessels.

Therefore, these two growth factors can be applied to the heart to elicit healing within the heart after a heart attack. The hydrogels keep the growth factors there and release them slowly so tat they can perform their healing magic.

Hopefully this experiment will lead to preclinical studies in larger animals (pigs and sheep), and then, hopefully, clinical trials in human patients.  See Salimath AS, et al., PLoS ONE 2012 7(11) e50980.