MicroRNAs as tiny regulators with big roles

Tiny RNAs, known as microRNAs, have been shown to regulate many components of the body’s cellular machinery. They are called microRNAs because they are only 22 nucleotides in size (compared to the 2200 nucleotide-long messenger RNA). Amazingly, these small non-coding RNAs can turn off the translation of their target genes. They act as control switches by targeting the 3′ untranslated regions of messenger RNAs (mRNA) for translational repression or cleavage, thus resulting in a reduction of protein levels. Because each microRNAs can regulate hundreds of messenger RNAs, there are probably few cellular processes not affected by microRNAs. For instance, microRNAs have recently emerged as playing important roles in a variety of cellular processes, such as heart development, stem cells, insulin secretion, and cholesterol synthesis. MicroRNAs were first discovered in worms more than 20 years ago. For many years, scientists thought that DNA was transcribed to RNA, and then translated to protein. Those proteins are major regulators in the cell. Now, they appreciate that there are more levels of control and a number of non-coding RNAs that regulate the level of cellular components. About one thousand microRNA genes have been discovered in the human genome. This makes the microRNAs one of the most abundant classes of regulatory genes. As a result of the discovery of this new and major level of regulation in the cell, Dr. Andrew Z. Fire and Dr. Craig C. Mello were awarded the 2006 Nobel Prize in Physiology or Medicine.

MicroRNA biogenesis

Unlike other RNAs, the production of microRNAs is quite different. As depicted in figure 1, the generation and activity of microRNAs requires special microprocessors, known as RNA polymerase II, Drosha, Exportin, Dicer, and RISC complex. RNA polymerase II transcribes (reads the microRNA DNA code) primary microRNA transcripts; then the Drosha process transforms primary microRNA into precursor microRNA in the nucleus. For activity and further processing, precursor microRNA are exported into cytoplasm by Exportin. In the cytoplasm, Dicer cuts precursor microRNA and generates mature 22 nucleotide long microRNA. Then, mature microRNA are incorporated into the RNA inducible silencing complex (RISC) where they target messenger RNAs (mRNA), either for degradation or translational repression. Even though there are extensive studies on microRNAs, it is still mostly unknown how microRNAs target specificity is determined and how they target messenger RNAs for mRNA degradation or translational repression. For a functional microRNA in the cell, it is amazing that a series of microprocessors should take place. They recognize different microRNAs as substrates and do their job as they are supposed to. It seems that the existence and regulation of microRNA processing abilities cannot be by mere chance.

MicroRNAs as therapeutics

MicroRNAs are considered “fine tuners” of cellular processes because of their subtle effects on their targets. However, because microRNAs can target a number of genes and genetic pathways, the study of microRNAs and their regulation and role in diseases is highly promising in terms of developing new therapeutic approaches. Treatments by targeting microRNAs using microRNA inhibitors (antisense RNA nucleotides) are under intense study and several of them have been shown to be effective in animal models. A MicroRNA known as miR-122, for instance, has been shown to regulate cholesterol levels. Scientists targeted this liver-specific microRNA by using a microRNA inhibitor and they found that the downregulation of miR-122 resulted in a 40% decrease in cholesterol levels in the blood.

MicroRNAs in cancer therapy

With the discovery of new and better tools to detect and manipulate microRNA levels in cell cultures and tissues, researchers are now attempting to identify the specific features of each microRNA and their role in cancer and other devastating diseases. There are some microRNAs that are highly correlated with cancer formation. Cancer is cellular anarchy characterized by a proliferation of cells without control. A group of miRNAs known as the miR-17-92 family have been found to increase, and their higher levels result in cancer formation as found in some lymphomas and solid tumors. It is believed that better understanding and use of microRNAs or microRNA inhibitors could enable doctors to treat diseases like cancer. In the near future, microRNA studies are also expected to provide early detection of progressive diseases, better markers for cancer initiation, and cancer specific drug selections.

MicroRNAs as cancer drug boosters

The most straightforward application of microRNA research has been cancer chemotherapies. The potential of use of microRNA applications to increase the effectiveness of current cancer drugs seems highly likely. Companies and universities are looking for microRNA partners to increase the effects of drugs like Taxol, which is currently used in chemotherapy. Taxol, for example, currently works for about 30% of lung cancer patients. But, if we can find a microRNA partner with that drug to make it 40%, it will mean saving thousands of lives. This is a hopeful sign for the future of cancer treatment. On the other hand, it is known that in the case of any chemotherapy, there are unwanted side effects. Although use of higher dose of drug will kill more tumors, the side effects of this drug will cause other issues. Discovery of partners like microRNAs that boost the effectiveness of cancer drugs or decrease side effects can help to treat more patients or help them overcome unwanted side effects.

Taking microRNAs to the heart of the matter

Heart diseases represent the primary cause of death in developed countries. Recent studies have identified microRNAs associated with heart diseases, including cardiac hypertrophy, heart failure (inability of the heart to pump sufficient blood to the organism), and myocardial infarction (the death of the cardiac muscle resulting from interruption of the blood supply). Mir-1 expression levels, for example, are low in human heart disease and it is known to regulate Hand2, a protein required for the growth of heart muscle cells. The levels of another microRNA, called miR-21, have consistently increased through cardiac stress and have been shown to regulate cardiac growth as well. Importantly, miR-133 is believed to repress cardiac hypertrophy, thus the use of synthetic miR-133 molecules is possible as a therapeutic for patients with pathological hypertrophy. However, more studies to understand heart-associated miRNAs are needed in order to have clinical trials for the treatment of heart diseases.

Figure 2. MicroRNAs in the heart. Recent studies have identified microRNAs that are associated with heart diseases, including arrhythmic heartbeat (Arrhythmias), cardiac hypertrophy (enlarged heart), septation defect, and cardiac muscle overgrowth (myocyte hyperplasia).

Micromanaging insulin secretion

MicroRNAs are also associated with the onset of diabetes. Diabetes affects about 23.6 million people in the United States. It can lead to serious health issues and even early death. Diabetes is marked by high levels of blood glucose (also called blood sugar). Complications of the disease are due to defects in insulin production and insulin action. Insulin is among the major regulators of sugar levels in the blood. The human genome contains a number of microRNA genes, whose functions are only beginning to come to light. One such microRNA, miR-375, is already implicated in the secretion of insulin from pancreatic cells, thus it represents a novel pharmacological target for the treatment of diabetes.

The mentioned cases above are examples of the tiny RNAs which regulate cellular processes. The loss of the control in such a small component of the cellular machinery can lead to serious problems, like cancer. To use a metaphor, the regular and healthy government of a state does not allow for the presence of multiple governors. Similarly, regulatory tiny RNAs require a controller who knows how the human body works at the macro and micro levels. This forces us to consider that whomever is controlling the human body must be all sustaining and all knowing. With each new scientific breakthrough, the wisdom of creation becomes more and more apparent. The field of miRNAs is a young research area. New discoveries about microRNAs have brought us new hopes for novel therapies to human diseases. However, future discoveries are required before these therapies can be used in a clinical setting.

Resources

• Qur’an: The Family of Imran 191 and The Cow 255.
• Caldas & Brenton. “Sizing up microRNAs as cancer genes”. Nature, 2005.
• Scott M. Hammond. “MicroRNA therapeutics: a new niche for antisense nucleic acids” Trends in Molecular Medicine, 2006.
• Rooij et al. “Toward MicroRNA–Based Therapeutics for Heart Disease” Circulation Research, 2008.
• National Diabetes Statistics, 2007. Retrived from http://diabetes.niddk.nih.gov/DM/PUBS/statistics/
• ScienceDaily. Not ‘Junk DNA’ After All: Tiny RNAs Play Big Role Controlling Genes. 2007.
• Callis & Wang. Taking microRNAs to heart. Trends in Molecular Medicine. 2008.
• Poy et al. A pancreatic islet-specific microRNA regulates insulin secretion. Nature,2004.
• Average mRNA length: B. Lewin, Genes 5, Table 2-2. Oxford University Press.
• MicroRNA biogenesis figure: http://content.nejm.org/content/vol359/issue25/images/large/14f1.jpeg

Matter&Beyond: What was your motivation for writing the book, The Heart-Mind Connection?

Well, that book was a collaboration with Dr. Ting who is a Cardiovascular Surgeon and it was actually his idea. He’s a very enlightened surgeon in that he, over the years of training in a practice as a cardiac surgeon, came to realize that the experience of undergoing cardiac surgery certainly taxed the mind and that the interaction of mind and heart was extremely important for outcomes. If you’re a cardiac surgeon and your goal is to improve your patients’ lives it became very clear to Dr. Ting that in order for that to happen cardiac surgeons and cardiologists had to be mindful of the fact that patients needed to be emotionally and cognitively intact, and so he asked me to collaborate on this book, and the book focused on the interaction between mood states and cardiac states.

M&B: What is the nature of this interaction? Does the heart affect the mind or does the mind affect the heart?

It’s bidirectional. There is an interaction or there is a relationship between mood disorders such as depression, anxiety, anger, and hostility and the actual human heart. The connection between those mood disorders and cardiac function is extremely important. But then also if you go into the experience of having heart disease and you were fortunate enough not to have one of those mood disorders, the experience of having cardiac disease will put you at risk for developing mood disorders. So it’s bidirectional. You can start out with heart disease and wind up having your health complicated by mood problems, or you can start out with mood problems and have your health complicated by heart disease. There’s an unholy alliance between depression and anxiety on the one hand and heart disease on the other.

M&B: What happens, let’s say, if you are someone who has a history of a major depressive disorder? What happens to his or her heart?

Major depressive disorder is a condition characterized by certain clear-cut symptoms, and those symptoms include having a depressed mood everyday most of the day for two weeks or more that includes losing pleasure in life, something we call anhedonia, not being able to take pleasure in normal activities that would normally make you happy. Along with that is loss of interest in things, guilt, or feelings of hopelessness, helplessness and worthlessness, loss of energy, loss of concentration, loss of appetite, sometimes even leading to weight loss, not being able to move the way you’re used to. So it’s sometimes you just curl up in bed and this is something we call psychomotor retardation or withdrawal. On the other hand sometimes depression makes you agitated so you can’t sit still and you’re running around sort of ringing your hands and so on. And then the final symptom of major depression is suicidality or morbid thinking. Always focused on death or wanting to die. So that’s the common sort of a cluster of symptoms we see in major depression. Now, say you have no history of heart disease but you suffer from major depressive disorder. You increase your risk of developing heart disease by having this condition of depression. So your risk of having a heart attack for example goes up like 1-1/2 to 2 times what it would be in someone without depression.

M&B: Is it similar with anger and hostility?

Some people believe that if you’re an angry person or a hostile person, that also increases your risk of developing depression. Say you have depression and you unfortunately have a heart attack. If you remain depressed then your risk of having another cardiac event, another minor cardiac infarction or a cardiac arrhythmia would go up three and a half times what it would be if you weren’t depressed. So that means that this connection between the brain and the heart, between depression and cardiac disease are tremendously important in terms of public health. Because those two diseases are causing most of the disability that exists, by the year 2020, the World Health Organization predicts that heart disease will be number one and depression will be number two in the entire world. That includes the developed world, the industrialized countries as well as the developing low income countries around the world. So in terms of the book that we did we thought it was important because these two illnesses, first of all feed off each other and cause a lot of heartache in the population but they also are the most important diseases in public health.

M&B: The heartbeat change is associated with love or certain joy. What is the physiology behind that?

Let me preface it by saying that this is the age old philosophical question which Plato and Aristotle sort of debated. Love is an idea as Plato thought. So, is there sort of an idea of beauty and of love that exists outside of the materialistic world? Are we somehow plugging into that ideal world of love as material organisms? Or does it come as a result, as an epiphenomenon of the material world we’re living? Does it emerge from the biological material world that we’re living? That in a way becomes a philosophical question. As scientists when we see human behavior, there’s something that we do, and it’s called reverse engineering. Let’s look at the behavior or the experience of human love. Where does that come from? Let’s work backwards from human love to try to understand in a biological way where it came from. We are the ultimate mammalian life. We might start a little controversy about that and especially with the all wars that we fight, but be that as it may, we are these high level, high functioning mammals. Our infants are born [in need of] altruism, which means that they are absolutely helpless when they are born. So parenting for the human being is extraordinarily important for the continuity of our species, and so our infants are in total need of care.

M&B: Isn’t this the same for all the animals?

It’s different in reptiles. If you go to Borneo and you study the life of the Komodo dragon, you’ll find out that komodo pups know immediately what they have to do when they plop out of komodo mommy. They have to roll around in their own feces in order to throw mommy off the scent because mommy will look at them as food. So that’s their strategy for avoiding being eaten by mommy. They also know how to climb up trees to get away from mommy. So here the survival strategy of reptiles is not one of attachment; it’s one of separation. All psychiatry is tied up in that simple fact. There would be no psychiatry if we were reptiles. But because we’re mammals, there’s psychiatry because all of the disorders we have are really disorders of a dysfunction in parental and social attachment. Only mammals for example have a part of the brain called the paralimbic cortex. So reptiles have primitive limbic areas, they have hippocampus, they have amygdala, so they show fear, and they act when they’re threatened, etc. They’re outfitted with those structures which allow them to do those simple attachments-attachments to food and to sexual objects etc., but don’t allow them to do social attachments very well, and there’s some reptiles that are better at it than others. Crocodiles, for example. They do a version of taking care of their pups. Birds also take care of their nestlings very well, and crocodiles actually have brains that are more kind of on the bird-like end of the spectrum, allowing them to do a better job as reptiles taking care of their young and having something of a social life. Nothing to the extent that mammals do and you can look at a mammal brain, and you can see where this is taking place. So the anterior cingulate is an area of extreme importance for mammalian behavior.

M&B: This unique mammal feature, the limbic system and the paralimbic system; would you elaborate a little bit more on them?

I had the privilege of meeting back in 1990 Paul McClain, who is a famous neuroscientist who coined the term limbic system. He studied emotional parts of the brain in reptiles and in monkeys and in human beings. He talked about what he called the mammalian behavioral triad. There are three things that mammals do that reptiles don’t do very well. One is that all mammalian infants cry for their mothers in the same way, so every lamb baas for its mother the same way. Every human infant cries for its mother in the same way, that’s called a separation cry, and you can study it. So when you look at rats they have an ultrasonic isolation cry; you can study it, and you can deprive them of relationships and you’ll see the cry. That’s typically mammalian. Another feature is play, so mammals play, and that serves a social binding function. We play sports together; it serves a social binding function. Then the third thing is maternal nurturance which is extremely important, and mammals do that parental nurturance piece pretty well.

M&B: Are there any experiments that may illustrate the role of love?

There are a couple of experiments that Paul did and they are very illustrative. They get us back to a focus on love. So one experiment was to take hamsters and you can ablate, sort of take out the anterior cingulate. When you do, the hamster pup no longer cries for its mother when it’s separated and the hamster mother no longer responds when the hamster pup cries for her. You can also put morphine in that area, and get pretty much the same results. So you could put morphine in the hamster pup’s brain in the anterior cingulated, and the hamster pup will no longer cry for their mother, it distorts that bonding. And you can do that with squirrel monkeys too and you’ll get similar results.

M&B: Do the results apply to human beings as well? Could the use of morphine stop a healthy heart or brain from giving love or responding to love?

One of the real problems that we face in psychiatry is when we run across an addicted mother. What happens to the mother child relationship when the mother is addicted to heroin or to cocaine or to other drugs of abuse? The child gets neglected, the mother no longer responds to the needs of the child. So we see there’s even a reflection of those basic experiments in the human model when we’re talking about maternal-child relationships. So again that’s kind of circumstantial evidence where there’s reverse engineering, and mothers also have this phenomenal hormone, neuropeptide hormone, this again is exclusively mammalian, called oxytocin. So it’s used in pregnant women to get them ready to give birth, and so once a pregnant woman gives birth it’s responsible for milk production and so on. This is another example of this – if we think about maternal love, one of the requirements for maternal love is that there needs to be oxytocin involved. Oxytocin needs to allow that brain to have the experience of being connected and bonded to offspring.

M&B: The relation between the brain and the other organs such as the heart, is it really a top down relation where the brain commands, or is it a bottom-up relation where bodily sensors form the human brain?

What you’re referring to when you bring up this fact that the body sends markers up to the brain, and the brain takes note of that and becomes behaviorally engaged, that is absolutely true, and it’s something that Tony Damasio calls a somatic marker hypothesis of how the brain works. This is also something that William James postulated in the late 1800s-that emotion is a reflection of the body’s activity. Your heart racing or your stomach turning, emotion is secondary, it’s the brain trying to make sense of what the body is going through. What came first, the chicken or the egg, that kind of a problem. The fact is, the organism works as a unity and you need all of its component parts for it to work, and you really can’t tease a part, what came first and what came second.

M&B: From our physiology, it is evident that we are born to love.

It is very spiritual about this kind of understanding of love and connecting it back to the brain. How the heart becomes a reflection of what the brain is about, and also this need we have as human beings for connection. Spirituality really is this need we seem to have as human beings to be connected to something greater than ourselves. So we have this part of our brain, the prefrontal cortex, which allows us to think into the future and when we think into the future, we gain security when we see a future of connectivity, of attachment. That becomes a workable definition of spirituality. So many of us feel connected to a higher power, to God or to nature, but that is also a common reflection of what people refer to as love, and again you can choose to understand its biological origins. The body will be connected up with that whole experience, so your heart will be connected up. If you’re feeling connected to God, or to the scene of an ocean, and you’re feeling connected to the universe, when you’re looking up at the stars, there will be a bodily sensation connected up with that experience, and that will become for you part of what you understand to be spirituality.

Heart regeneration in mammals

Such heart regeneration holds the promise for the treatment of heart failure following heart attacks. But so far, the adult human heart is known not to show adequate regeneration or replacement of dead tissue with functional tissue such as beating cardiomyocytes (cardiac muscles) and arteries. When a patient has successive heart attacks and myocardial infarctions (death of cardiac muscle resulting from interruption of the blood supply), the number of dead cells increases due to the decreased level of oxygen reaching the heart tissue. That’s one of the reasons heart disease is so deadly.

The rates of cardiac regeneration, from fish to amphibians to mammals, demonstrates a decreasing trend — high in fish, moderate in amphibians, and limited in mammals. The regeneration mechanism is thought to occur via incorporating stem cells, using differentiation into cardiac muscle and other cell types, or via dedifferentiation of cardiomyocytes. It is known that the heart of an adult zebra fish can regenerate without scar formation, whereas adult rodents and humans respond with a fibrous scar, without obvious cardiomyocyte regeneration. This remarkable phenomenon had been demonstrated in other fish and amphibians, but never before in a mammal. Recently, researchers at UT Southwestern Medical Center showed that a newborn mouse’s heart can fully heal itself.

Sadek’s group at UT Southwestern Medical Center at Dallas showed that the mammalian heart demonstrates a temporary regeneration capacity in newborn mice. After slowing down the body functions by cooling the body of a mouse, they performed a very delicate heart surgery, removing about 15 percent of the apex of a 1-day-old newborn mouse heart. Within a short period (three weeks), they showed that heart had healed and the function of heart had returned to normal. But when mice are a week old, this remarkable ability of regeneration disappears, and damage to the heart results in the thinning of the heart wall at the site of injury, and the loss of the pumping capacity of heart, also known as heart failure. There seems to be a barrier to regeneration after 7 days. This 7-day window in mice could correspond to a few months after birth in humans. Several reports suggest that human heart may also have some ability to regenerate in infancy.

If newborn animals and infants are able to regenerate their hearts, there could be ways to remind the heart how do this or restart this ability in adulthood to allow regeneration in a broader window. Could there be means to induce regeneration by gene therapy, using small molecules, drugs or hormones? This new discovery brings new approaches to study heart disease and hopes that one day, heart disease — the number one killer in the world — could be treated. More studies are needed and a number of labs have already started to invest in this new model of heart regeneration.

Human heart cell turnover and regeneration

The heart is the least regenerative organ in our body. Once cardiomyocytes are damaged through heart attacks, the heart heals by scar formation instead of regeneration. This results in a loss of contractile function and often ends in heart failure. Lack of regeneration in an adult heart is associated with the complexity and inability of cardiomyocytes to divide, along with the absence of adequate muscle-producing cardiac stem cells in the heart.

Cardiomyocytes proliferate extensively during embryonic development but slow dramatically around birth. The growth of heart continues after birth through the increase in cardiomyocyte size, known as hypertropy. This allows DNA synthesis and nuclear division and results in binucleated cardiomyocytes.

Increasing evidence strongly suggests that the human heart shows a degree of cardiomyocyte repopulation (introduction of new cardiomyoctes). It is always challenging to study human heart cellular homeostasis, as it is limited in the availability of human samples and the means to work on it. Who knew nuclear testing during the Cold War would help to uncover dynamics of human cardiomyocyte turnover? Using a technique based on radiocarbon dating of DNA with carbon-14, released from nuclear tests, Bergmann and his colleagues from the Karolinska Institute in Sweden showed that the cardiomyocyte turnover rate is about 1 percent per year at age 20, with a decline to 0.4 percent per year at age 75. This is based on the idea that people born during nuclear tests following World War II until the Limited Nuclear Test Ban Treaty (1963), any cardiomyocyte repopulation should result in lower carbon-14 concentrations. These findings imply that around age 50, about half of the cardiomyocytes in the human heart are generated after birth. However, another study puts emphasis on the importance of cell deaths (apoptosis) for heart cell turnover, asserting that these rates could be much higher (7-40 percent per year). Those findings bring new hopes to heart disease. If the repopulation potential of heart could be therapeutically targeted, the rate of turnover could be extended to overcome the inability to recover cardiomyocyte loss and cardiac contractility after heart attacks.

The better regenerative capacity of fish and amphibians, compared to that of mammals, seems to stem from the presence of species-specific differences. It has been suggested that the limited regeneration potential of mammalian hearts following injury increases survival by prioritizing homeostasis and fibrosis (scar formation by excess connective tissue). Bleeding from the heart in a high-pressure circulation probably favors the more rapid fibrous healing, instead of regeneration, whereas small animals have a low-pressure circulatory system and oxygenation isn’t needed all the time. This phenomenon probably applies to the regeneration of the newborn mouse heart, which also made the removal of the apex of the newborn mouse heart possible.

Cardiac stem cells for regeneration

The heart is a mosaic of various cell types including valvular, arterial, smooth muscle, pacemaker, endothelial, autonomic ganglia, fibroblasts and cardiomyocytes. Those cells have essentially the same genetic makeup but they show a great diversity. Could there be a common cardiac stem cell that gives rise to all those cell types in the heart? There are a number of studies suggesting the presence of such stem cells, though why they fail to regenerate the heart following heart attacks remains unknown.

There have been a number of attempts to discover cardiac stem cells. Some stem cells have been studied in animals and even considered as possible therapies in human trials. Sources of those stem cells could be classifies as resident and non-resident (exogenous) cells of heart. Exogenous stem cell types include skeletal myoblasts, hematopoietic stem cells, mesenchymal stem cells from bone marrow and circulating endothelial cells. Many approaches to identify resident cardiac stem cells are based on knowledge from hematopoietic stem cells. Using surface proteins on the cells known to enrich bone marrow stem cells, several types of resident stem cells are shown to exist in the heart. There are limited improvements in cardiac function using those cells, but the benefits of those cells are thought to be through other mechanisms instead of replacement of dead tissue in the damaged heart.

A study demonstrating the renewal of a newborn mice heart does not completely rule out resident cardiac stem cells as a source of new beating heart cells, but points out the likelihood of their originating from cardiomyocytes by dedifferentiation. Along with a number of attempts to treat heart failure by using stem cells, recent findings offer hope that researchers and doctors will one day able to cure heart disease. Knowing that “there is no disease that God has created, except that He also has created its treatment,” our duty is to study hard and to develop new technologies to find the prospective treatments for heart failure to serve humanity.

References

• Porrello et al. 2011. “Transient Regenerative Potential of the Neonatal Mouse Heart.” Science 25 February: 1078–1080.
• Bergmann et al. 2009. “Evidence for Cardiomyocyte Renewal in Humans.” Science, 3 April: 98–102.
• Charles E. Murry and Richard T. Lee. 2009. “Turnover after the fallout.” Science, V324.
• O.Bergmann et al. 2009. Science 324, 98.
• Simonetta Ausoni and Saverio Sartore. 2009. “From fish to amphibians to mammals: in search of novel strategies to optimize cardiac regeneration.” JBC. 184 (3).
• Martin-Puig et al. 2008. “Lives of a hear cell: Tracing the origins of cardiac progenitors.” Cell Stem Cell 2. April.
• Nevada Nuclear Testing Site: http://mason.gmu.edu/~kcherrix/atomichome.html
• Sahih al-Bukhari, Vol. 7, Book 71.