Repairing a Heart with MiRNA

This week, scientists in Italy have discovered a genetic trigger that can cause heart muscle cells to start dividing to produce new cells and to repair the damage done by a heart attack.  Mauro Giacca is at the International Centre for Genetic Engineering and Biotechnology in Trieste...

Chris - Mauro, why don’t heart cells normally divide?

Mauro -   The reason why they don’t divide is not known, but it is a matter of fact that after birth, there is just a short window of a few weeks for cell division and then cells in the heart stop dividing and the capacity of their proliferation over their whole life remains very, very limited.

Chris -   In other words, if you actually try to make the heart cells divide by injuring it, in the same way that if you cut your skin, the skin will grow, heart muscle doesn't regrow.

Mauro -   Heart muscle doesn’t regrow and repair. Normally, what takes place is a scarring mechanism.  So eventually, after for example a myocardial infarction, the end result is the formation of a scar and the regeneration of the tissue.

Chris -   And if you have that scar, what are the functional consequences for the patient?

Mauro -   The problem with a scar is that over time, the heart remodels in a negative manner and the pumping function of the organ is progressively impaired.  This is a condition that clinically is known as heart failure. 

Chris -   So, the obvious thing is, if you could work out how to make the normal healthy heart cells regrow so that a heart attack or some similar injury would heal with the replacement of muscle rather than fibrous scar tissue that doesn’t have any pumping function, then people are much more likely to have a good outcome.  So, what can we actually do to realise that possibility?

Mauro -   One possibility is to find the trigger to push cardiomyocytes to divide again. This is genetically possible also because other species like the salamander or the fish are known to regenerate completely the heart after the organ is damaged.  So we, as mammals have lost this possibility during evolution probably because scarring is a much faster mechanism over cardiac repair and allows the animals to survive.

Chris -   So, do you have any insights into why the cells are locked into these non-dividing state and what we could do to unlock them so they can begin to divide again?

Mauro -   I believe nobody knows why they're locked into this non-dividing state, but what we know is that – what we did was to see if we could unlock this mechanism by treating these cells with microRNAs - that is to try genetically to switch those on and proliferate in this state. 

MicroRNAs are small stretches of RNA which are normally produced by our genome.  There are about a couple of thousand genes coding for microRNAs and each of these target tens or hundreds of different genes simultaneously.  They downregulate these genes and so, we thought that this might be a way to find microRNAs that could stimulate cardiac myocytes proliferation. 

We did this in a robotic format – that is we screened almost 1,000 human microRNAs for their capacity to extend the proliferation capacity of neonatal rodent cardiomyocytes.  We did these first in rats and then in mice, and then eventually, we found 40 microRNAs that work in these two species and also work in human cardiomyocyte cells.

Chris -   So, these microRNAs, they're short sequences of genetic material which can effectively switch off other genes and your reasoning is that there may be some microRNAs in a cardiac muscle cell that switch off the ability of that cell to divide.  So, by putting in a different microRNA, you could either switch on a function that makes the cells divide or rule out or negate the effect of these other microRNAs so that the cells can begin to divide.

Mauro -   This is exactly the case – in fact, we see that several of the 40 microRNAs that we found from our screen are those that are normally active, so expressed at high levels during the embryonic and foetal life and then they are switched off when the cells stop dividing after birth.  So we believe that they play a role in maintaining the actual proliferation of cardiomyocytes during the prenatal period.

Chris -   And added to cells in the dish, they make the cells grow so you're obviously on the right track.  What about when you put them into a real animal?  Do they produce all kinds of effects throughout the body or are they fairly discrete in their effects on just the animal’s heart?

Mauro -   The best ones in triggering cardiomyocyte proliferations appear to be rather restricted to cardiac cells which is good news because when you think of proliferation, immediately what comes to your mind is an unwanted effect of triggering proliferation of other cell types. 

They're quite specific for cardiomyocyte cells and when we inject them in vivo in neonatal animals, they make the heart grow bigger.  So you see a much bigger heart with plenty of replicating cells in the ventricles.  However, these hearts are, as far as we can say by echocardiography, they are perfectly functional.

The most exciting finding was that when we had these microRNAs embedded into a viral vector for efficient delivery into the heart and injected into the region bordering a myocardial infarction.  These were capable of entering the cells and convincing the residual cardiomyocyte cells to proliferate.  So the infarct was repaired and not through a scarring mechanism, but largely by regeneration of the contractile cells.  The cardiac function of the infarcted animal treated with the microRNAs was almost similar to the cardiac function of an untreated animal and so, the animal which was non-infarcted.

Chris -   Do you know whether or not the virus was targeting cardiomyocytes themselves and making them grow or was it in some way targeting other cells and then making those other cells make the cardiomyocytes grow?  Which of those two is it?

Mauro -   That’s a good question but I think we know the answer because we have gained a lot of experience with the use of this particular kind of vector.  These are vectors based on the adeno-associated virus, AAV and these vectors have an exquisite capacity to target post-mitotic cells  – in this case, cardiomyocytes.  And not fibroblasts or endothelial cells which are the two other major contributing cells in the heart.  So we are quite sure that the effect is triggered by an action direct in the cardiomyocytes.