Usually, each cell has exactly one nucleus. But the cells of our skeletal muscles are different: These long, fibrous cells have a comparatively large cytoplasm that contains hundreds of nuclei. But up to now, we have known very little about the extent to which the nuclei of a single muscle fiber differ from each other in terms of their gene activity, and what effect this has on the function of the muscle.

A team led by Professor Carmen Birchmeier, head of the research group on Developmental Biology / Signal Transduction at the Max Delbrueck Center for Molecular Medicine in the Helmholtz Association (MDC), has now unlocked some of the secrets contained in these muscle cell nuclei. As the researchers report in the journal Nature Communications, the team investigated the gene expression of cell nuclei using a still quite novel technique called single-nucleus RNA sequencing — and in the process, they came across an unexpectedly high variety of genetic activity.

Muscle fibers resemble entire tissues

“Due to the heterogeneity of its nuclei, a single muscle cell can act almost like a tissue, which consists of a variety of very different cell types,” explains Dr. Minchul Kim, a postdoctoral researcher in Birchmeier’s team and one of the two lead authors of the study. “This enables the cell to fulfill its numerous tasks, like communicating with neurons or producing certain muscle proteins.”

Kim undertook the majority of the experimental work in the study, and his data was also evaluated at the MDC. The bioinformatics analyses were performed by Dr. Altuna Akalin, head of the Bioinformatics and Omics Data Science Platform at the MDC’s Berlin Institute of Medical Systems Biology (BIMSB), and Dr. Vedran Franke, a postdoctoral fellow in Akalin’s team and the study’s co-lead author. “It was only thanks to the constant dialogue between the experiment-based and theory-based teams that we were we able to arrive at our results, which offer important insight for research into muscle diseases,” emphasizes Birchmeier. “New techniques in molecular biology such as single cell sequencing create large amounts of data. It is essential that computational labs are part of the process early on as analysis is as important as data generation,” adds Akalin.

Injured muscles contain activated growth-promoting genes

The researchers began by studying the gene expression of several thousand nuclei from ordinary muscle fibers of mice, as well as nuclei from muscle fibers that were regenerating after an injury. The team genetically labeled the nuclei and isolated them from the cells. “We wanted to find out whether a difference in gene activity could be observed between the resting and the growing muscle,” says Birchmeier.

And they did indeed find such differences. For example, the researchers observed that the regenerating muscle contained more active genes responsible for triggering muscle growth. “What really astonished us, however, was the fact that, in both muscle fiber types, we found a huge variety of different types of nuclei, each with different patterns of gene activity,” explains Birchmeier.

Stumbling across unknown nuclei types

Before the study, it was already known that different genes are active in nuclei located in the vicinity of a site of neuronal innervation than in the other nuclei. “However, we have now discovered many new types of specialized nuclei, all of which have very specific gene expression patterns,” says Kim. Some of these nuclei are located in clusters close to other cells adjacent to the muscle fiber: for example, cells of the tendon or perimysium — a connective tissue sheath that surrounds a bundle of muscle fibers.

“Other specialized nuclei seem to control local metabolism or protein synthesis and are distributed throughout the muscle fiber,” Kim explains. However, it is not yet clear what exactly the active genes in the nuclei do: “We have come across hundreds of genes in previously unknown small groups of nuclei in the muscle fiber that appear to be activated,” reports Birchmeier.

Muscle dystrophy seemingly causes many nuclei types to be lost

In a next step, the team studied the muscle fiber nuclei of mice with Duchenne muscular dystrophy. This disease is the most common form of hereditary muscular dystrophy (muscle wasting) in humans. It is caused by a mutation on the X chromosome, which is why it mainly affects boys. Patients with this disease lack the protein dystrophin, which stabilizes the muscle fibers. This results in the cells gradually dying off.

“In this mouse model, we observed the loss of many types of cell nuclei in the muscle fibers,” reports Birchmeier. Other types were no longer organized into clusters, as the team had previously observed, but scattered throughout the cell. “I couldn’t believe this when I first saw it,” she recounts. “I asked my team to repeat the single-nucleus sequencing immediately before we investigated the finding any further.” But the results remained the same.

The mouse nuclei resemble those of human patients

“We also found some disease-specific nuclear subtypes,” reports Birchmeier. Some of these are nuclei that only transcribe genes to a small extent and are in the process of dying off. Others are nuclei that contain genes that actively repair damaged myofibers. “Interestingly, we also observed this increase in gene activity in muscle biopsies of patients with muscle diseases provided by Professor Simone Spuler’s Myology Lab at the MDC,” says Birchmeier. “It seems this is how the muscle tries to counteract the disease-related damage.”

“With our study, we are presenting a powerful method for investigating pathological mechanisms in the muscle and for testing the success of new therapeutic approaches,” concludes Birchmeier. As muscular malfunction is also observed in a variety of other diseases, such as diabetes and age- or cancer-related muscle atrophy, the approach can be used to better research these changes too. “We are already planning further studies with other disease models,” Kim confirms.

The evolution of male ornaments has therefore been fascinating to biologists since ever. Already Charles Darwin wondered of how such exaggerated, energy-consuming and in principle harmful structures could have been created by natural selection. Using the example of the swordtail fish (Xiphophorus hellerii), he explained his theory of sexual selection. Darwin’s basic idea: If females prefer to mate with the carriers of striking ornamental traits, such traits might become established in the course of evolution even though they are likely to be harmful for their owners.

Scientists from Würzburg, Constance and the USA have now been successful in finding the genetic bases of this evolutionary model in Xiphophorus, also well known to aquarists as one of their favourite pets. Among all eligible genes, the researchers identified some that are responsible for the development of the corresponding ornamental trait in this species of fish. Their findings also suggest that in the swordtail a gene that is actually important for neuronal processes in the brain has taken on an additional new function during evolution.

The scientists published their findings in the journal Current Biology. Manfred Schartl, a senior professor at the Department of Developmental Biochemistry at the University of Würzburg, is the lead author of the study. The project was co-initiated by the evolutionary biologist Axel Meyer from Constance University with whom the Würzburg researchers have been investigating this phenomenon for many years. For more than two decades, the two laboratories at the Universities of Würzburg and Constance have jointly researched the genetic basis of the sword. The current study has now brought the scientists a big step closer to understanding the genetic basis of the extended caudal fin of swordtails.

“In several species of the genus Xiphophorus, the males carry a so-called ‘sword’, a striking extension of the lower edge of the tail fin, which is yellow, orange or red in colour and surrounded by a dark black margin,” explains Manfred Schartl. The sword develops during puberty and can be as long as the fish itself in some species. This should actually be a disadvantage, because the conspicuous body ornament attracts predators on the one hand and on the other hand makes escaping more difficult as it reduces swimming performance. However, the females of Xiphophorus hellerii and several related species prefer to mate with males that carry a long sword — males with shorter swords literally lose out in this competition.

Gradually excluding the suspects

The genetic bases of this extension of the caudal fin in Xiphophorus have previously been unknown. However, knowledge of this phenomenon is necessary to test hypotheses about the role of sexual selection at the molecular genetic level.

The scientists took a gradual approach to pinpointing the responsible genes. They started by looking for all genes that are specifically active in the sword developing part of the tail fin, but not in fin regions that do not form a sword. “This process resulted in a set of 329 differentially expressed genes in all sword transcriptomes,” said Schartl, describing the result. The term transcriptome refers to the entirety of genes that are transcribed in a cell or tissue at a certain point in time, i.e. are active.

The consideration that genes responsible for sword formation are only expressed in males led to a significant reduction in the number of suspects in the next step. The scientists created transcriptomes of cells from specific areas of the caudal fins in both male and female specimens. If the females showed comparable activities to males, it was clear that these genes are not among the sought-after candidates. After this process, 255 of the original 329 genes remained.

“Interestingly, this comparison revealed that a spatial pattern of five transcription factors — Zic1, Hoxb13a, Six2a, Tbx3a and Pax9 — is responsible for organising the preconditions in the caudal fin for the development of a sword, and that this pattern is also present in females,” said Schartl.

Backcrossings provide important information

Genetic mapping came next to further reduce the still high number of 255 candidate genes. For this purpose, swordtail males were crossed with females of a related species whose males had lost their sword in the course of evolution. The male descendants from this mating have swords of different lengths due to the mixing of the parental genomes depending on their random genetic make-up. Sequencing those genomes using special high-throughput techniques then made it possible to correlate certain chromosome segments with sword formation, and those with the list of candidate genes. Finally, three genes were left over.

Spotlight on one main suspect

The gene with the scientific name kcnh8 proved to be crucial for the development of the male characteristic. “This gene codes for a potassium channel — a group of channels that play an important role in particular in the transmission and processing of stimuli in the nervous system,” said Schartl. The new findings point to a gene with a primary function in neural cells that was recruited during evolution for developing the male sword about three to five million years ago, i.e. early during the diversification of swordtail fishes. The new function is not due to structural changes within the gene and its product, but to changes in gene regulation.

Indeed, experiments show that kcnh8 in the sword during normal development and after treatment with male hormones is highly upregulated in the region where the sword is organised. In all other fin areas of the males and in female caudal fins it is only weakly expressed. In addition, further studies show a direct correlation between the level of gene expression of kcnh8 and the length of swords.

Schartl and Meyer received support for their research on potassium channels from an expected source: botanists from the University of Würzburg. Rainer Hedrich, who heads the Department of Molecular Plant Physiology and Biophysics, and his colleague, Professor Dietmar Geiger, have been studying potassium channels for a long time. The techniques they used — special patch-clamping methods — could be easily transferred from plants to the fish.

Potassium channels transport electrically charged particles and thus cause changes in the membrane potential in cells and tissues. According to the scientists, such channels create tissue-wide bioelectric gradients which affect the overall structure of the cellular microenvironment. Similar phenomena have been observed in the proliferation of cancer cells and have led to hypotheses about the importance of ion gradients for growth control. The role of Kcnh8 in the development of the ventral caudal outgrowth in male swordtails is in good agreement with these models.

The bacterial population in the gut, known as the gut microbiota, is the largest reservoir of bacteria in the body. Research has increasingly shown that the host and the gut microbiota are an excellent example of systems with mutually beneficial interactions. Recent observations also revealed a link between mood disorders and damage to the gut microbiota. This was demonstrated by a consortium of scientists from the Institut Pasteur, the CNRS and Inserm, who identified a correlation between the gut microbiota and the efficacy of fluoxetine, a molecule frequently used as an antidepressant. But some of the mechanisms governing depression, the leading cause of disability worldwide, remained unknown.

Using animal models, scientists recently discovered that a change to the gut microbiota brought about by chronic stress can lead to depressive-like behaviors, in particular by causing a reduction in lipid metabolites (small molecules resulting from metabolism) in the blood and the brain.

These lipid metabolites, known as endogenous cannabinoids (or endocannabinoids), coordinate a communication system in the body which is significantly hindered by the reduction in metabolites. Gut microbiota plays a role in brain function and mood regulation

Endocannabinoids bind to receptors that are also the main target of THC, the most widely known active component of cannabis. The scientists discovered that an absence of endocannabinoids in the hippocampus, a key brain region involved in the formation of memories and emotions, resulted in depressive-like behaviors.

The scientists obtained these results by studying the microbiotas of healthy animals and animals with mood disorders. As Pierre-Marie Lledo, Head of the Perception and Memory Unit at the Institut Pasteur (CNRS/Institut Pasteur) and joint last author of the study, explains: “Surprisingly, simply transferring the microbiota from an animal with mood disorders to an animal in good health was enough to bring about biochemical changes and confer depressive-like behaviors in the latter.”

The scientists identified some bacterial species that are significantly reduced in animals with mood disorders. They then demonstrated that an oral treatment with the same bacteria restored normal levels of lipid derivatives, thereby alleviating the depressive-like behaviors. These bacteria could therefore serve as an antidepressant. Such treatments are known as “psychobiotics.”

“This discovery shows the role played by the gut microbiota in normal brain function,” continues Gérard Eberl, Head of the Microenvironment and Immunity Unit (Institut Pasteur/Inserm) and joint last author of the study. If there is an imbalance in the gut bacterial community, some lipids that are vital for brain function disappear, encouraging the emergence of depressive-like behaviors. In this particular case, the use of specific bacteria could be a promising method for restoring a healthy microbiota and treating mood disorders more effectively.

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In order to formulate effective theories in physics, microscopic details are set aside in favour of macroscopically observable quantities. A cup of water can be described by properties like pressure, temperature and density of the fluid, whereas the position and velocity of the individual water molecules are irrelevant. A phase transition describes the change of a macroscopic system from one state of matter, like fluid, to a different state of matter, like gaseous. The properties of macroscopic systems — so-called many-body systems — can be described as emergent because they result from the interaction of individual components which themselves do not possess these properties.

“I have long been interested in how this dramatic macroscopic change at a phase transition emerges from the microscopic description,” states Selim Jochim. To answer this question, the researchers designed an experiment in which they assembled a system from individual ultracold atoms. Using this quantum simulator, they investigated how collective behaviour arises in a microscopic system. To this end, they trapped up to twelve atoms in a tightly focused laser beam. In this artificial system it is possible to continuously tune the interaction strength between the atoms from non-interacting to being the largest energy scale in the system. “On the one hand, the number of particles in the system is small enough to describe the system microscopically. On the other hand, collective effects are already evident,” explains Luca Bayha, a postdoc in Prof. Jochim’s team.

In their experiment, the Heidelberg physicists configured the quantum simulator such that the atoms attract one another, and if the attraction is strong enough, form pairs. These pairs of atoms are the necessary ingredient for a phase transition to a superfluid — a state in which the particles flow without friction. The current experiments focused on when the pair formation emerges as a function of the interaction strength and the particle number. “The surprising result of our experiment is that only six atoms show all the signatures of a phase transition expected for a many-particle system,” adds Marvin Holten, a doctoral student in Prof. Jochim’s group.

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As they began to comprehend the depths of diversity across the planet, they noticed an interesting pattern. The number of species increases drastically from the poles to the equator. This phenomenon, known as the latitudinal gradient of species diversity, has helped define the tropics as home to most of the world’s biodiversity. Scientists estimate that tropical forests contain more than half the species on earth, from plants and insects to birds, amphibians, and mammals.

These biologically rich areas are known as biodiversity hotspots. To qualify as a hotspot, a region must have at least 1,500 vascular plants species occurring nowhere else and have 30 percent or less of its original natural vegetation. In other words, it must be both irreplaceable and threatened.

While scientists agree that most biological diversity originated in the tropics, the jury is still out on how tropical species diversity formed and how it is maintained. A new study published in Science addresses these long-standing questions.

In “The evolution of tropical biodiversity hotspots,” researchers argue that tropical species form faster in harsh species-poor areas but accumulate in climatically moderate areas to form hotspots of species diversity. Drawing on decades of expeditions and research in the tropics and the scientists’ own knowledge and sampling of tropical bird diversity, the research team assembled a large and complete phylogenomic dataset for a detailed investigation of tropical diversification.

“This is our magnum opus,” said Elizabeth Derryberry, associate professor in the University of Tennessee, Knoxville’s Department of Ecology and Evolutionary Biology (EEB) and a senior author of the study. “This research is the product of a decades-long international collaboration to produce a completely sampled evolutionary history of a massive tropical radiation — the 1,306 species of suboscine passerine birds.”

Roughly one in three Neotropical bird species is a suboscine, making it the predominant avian group in the Neotropic terrestrial habitat — which ranges from the Andes snow line to the Amazon lowlands — and a perfect group to shed light on the origins of tropical biodiversity.

“The tropics are a natural laboratory for speciation research,” said Michael Harvey, a recent EEB postdoctoral student and lead author of the study. “Many high-profile studies over the years sought answers to fundamental questions concerning species formation and maintenance.” These earlier projects, he added, sampled only a minority of the existing species within the group being studied. In addition, said Derryberry, data analysis limitations in nearly all of the previous studies left them open to estimation errors.

For this study, Derryberry, Harvey, EEB Professor Brian O’Meara, and fellow researchers used a time-calibrated phylogenomic tree to provide information needed for estimating the dynamics of suboscine diversification across time, lineages, and geography. They also used the tree to test links between the dynamics and potential drivers of tropical diversity.

“We took no shortcuts in this study,” Derryberry said. “We leveraged this unparalleled sampling of tropical diversity to illustrate the tempo and geography of evolution in the tropics. It is the first study to demonstrate conclusively that tropical biodiversity hotspots are linked to climates that are both moderate and stable.”

The team discovered that species-rich regions in the tropics contain diversity accumulated during a protracted evolutionary period. A key result of their study is that the best predictor of elevated speciation rates in North and South American suboscines is low species diversity. In other words, new species form at higher rates in areas containing relatively few species.

“The qualities that nurture diversity, lower extinction, and promote the gradual accumulation of species are, paradoxically, not the ones that support biodiversity hotspots,” Harvey said. “The hotspots are seeded by species born outside the hotspot in areas characterized by more extreme and less climatically stable climates.”

The team discovered that, overall, extreme environments limit species diversity but increase opportunities for populations to evolve to become distinct species. Moderate climates, on the other hand, limit speciation but provide more opportunities for species diversity to accumulate.

“Our study will pave the way for future investigations of evolution in the world’s diversity hotspots,” Derryberry said. “This paper marks not only a change in our understanding of evolution in the tropics, but also in acknowledgement and valuation of the diversity of culture, expertise, and perspective in the field of ornithology.”

International collaboration for the study included researchers from Colombia, Brazil, Uruguay, and Venezuela as well as ornithologists from groups underrepresented in the sciences, include Latino and women researchers.

“Our research on rats with heart failure shows that exercise reduces the severity of the disease, improves heart function and increases work capacity. And the intensity of the training is really importance to achieve this effect,” says Thomas Stølen, a researcher at the Norwegian University of Science and Technology (NTNU).

Stølen and his colleague Morten Høydal are the main authors of a comprehensive study published in the Journal of Molecular and Cellular Cardiology. The researchers went to great lengths to investigate what happens inside tiny heart muscle cells after regular exercise.

“We found that exercise improves important properties both in the way heart muscle cells handle calcium and in conducting electrical signals in the heart. These improvements enable the heart to beat more vigorously and can counteract life-threatening heart rhythm disorders,” says Stølen.

For a heart to be able to beat powerfully, regularly and synchronously, a lot of functions have to work together. Each time the heart beats, the sinus node — the heart’s own pacemaker — sends out electrical impulses to the rest of the heart. These electrical impulses are called action potentials.

All the heart muscle cells are enclosed by a membrane. At rest, the electrical voltage on the inside of the cell membrane is negative compared to the voltage on the outside. The difference between the voltage on the outside and the inside of the cell membrane is called the resting membrane potential.

When the action potentials reach the heart muscle cells, they need to overcome the resting membrane potential of each cell to depolarize the cell wall. When this happens, calcium can flow into the cell through channels in the cell membrane.

Calcium initiates the actual contraction of the heart muscle cells. When this process is complete, calcium is transported out of the cell or back to its storage site inside each heart muscle cell. From there, the calcium is ready to contribute to a new contraction the next time an action potential comes rushing by.

If the heart’s electrical conduction or calcium management system fails, the risk is that fewer heart muscle cells will contract, the contraction in each cell will be weak, and the electrical signals will become chaotic so that the heart chambers begin to flutter.

“All these processes are dysfunctional when someone has heart failure. The action potentials last too long, the resting potential of the cells is too high, and the transport function of the calcium channels in the cell wall is disturbed. Calcium then constantly leaks from its storage places inside every heart muscle cell,” Stølen says.

Before Stølen gives us the rest of the good news, he notes, “Our results show that intensive training can completely or partially reverse all these dysfunctions.”

Normally, the sinus node causes a human heart to beat between 50 and 80 beats every minute when at rest. This is enough to supply all the organ systems and cells in the body with as much oxygen-rich blood as they need to function properly.

When we get up to take a walk, our heart automatically starts beating a little faster and pumping a little harder so that the blood supply is adapted to the increased level of activity. The higher the intensity of the activity, the harder the heart has to work.

Exercise strengthens the heart so it can pump more blood out to the rest of the body with each beat. Thus, the sinus node can take it a little easier, and well-trained people have a lower resting heart rate than people who have not done regular endurance training.

At the other end of the continuum are people with heart failure. Here the pumping capacity of the heart is so weak that the organs no longer receive enough blood to maintain good functioning. People with heart failure have a low tolerance for exercise and often get out of breath with minimal effort.

In other words, increasing the pumping power to the heart is absolutely crucial for the quality of life and health of people with heart failure.

Many of the more than 100,000 Norwegians who live with heart failure have developed the condition after suffering a major heart attack — just like the rats in Stølen and Høydal’s study.

In the healthy rats, the heart pumped 75 percent of the blood with each contraction. In rats with heart failure, this measure of pump capacity, called ejection fraction, was reduced to 20 per cent, Stølen says.

The ejection fraction increased to 35 percent after six to eight weeks with almost daily interval training sessions on a treadmill. The rats did four-minute intervals at about 90 percent of their maximum capacity, quite similar to the 4 × 4 method that has been advocated by several research groups at NTNU for many years.

“The interval training also significantly improved the rats’ conditioning. After the training period, their fitness level was actually better than that of the untrained rats that hadn’t had a heart attack,” says Stølen.

Impaired calcium handling in a heart muscle cell not only causes the cell to contract with reduced force every time there is an action potential. It also causes the calcium to accumulate inside the fluid-filled area of the cell — the cytosol — where each contraction begins.

The calcium stores inside the cells are only supposed to release calcium when the heart is preparing to beat. Heart failure, however, causes a constant leakage of calcium out of these stores. After each contraction, calcium needs to be efficiently transported back into the calcium stores — or out of the heart muscle cell — via specialized pumps. In heart failure patients, these pumps work poorly.

When a lot of calcium builds up inside the cytosol, the heart muscle cells can initiate new contractions when they’re actually supposed to be at rest. An electrical gradient develops which causes the heart to send electrical signals when it shouldn’t. This can cause fibrillation in the heart chambers. This ventricular fibrillation is fatal and a common cause of cardiac arrest.

“We found that interval training improves a number of mechanisms that allow calcium to be pumped out of the cells and stored more efficiently inside the cells. The leakage from the calcium stores inside the cells also stopped in the interval-trained rats,” says Stølen.

The effect was clear when the researchers tried to induce ventricular fibrillation in the diseased rat hearts: they only succeeded at this in one of nine animals that had completed interval training. By comparison, they had no problems inducing fibrillation in all the rats with heart failure who had not exercised.

So far, the research group had shown that exercise improves calcium management in diseased heart muscle cells in several ways. The training also makes the electrical wiring system of the heart more functional.

In addition, they showed that exercise counteracted processes that cause the heart to become big and stiff.

Taken together, these improvements make each heartbeat more powerful and reduce the severity of heart failure. The risk of dangerous ventricular fibrillation was also reduced.

But Stølen and team still lacked an answer to why exercise corrects slow action potentials and ensures that the heart muscle cells are able to take care of calcium in the right way.

Therefore, they investigated whether the training had altered the genetic activity inside the rat cells. Thousands of different types of micromolecules called micro-RNA probably control most of this activity through direct interaction with genes.

“It turned out that 55 of the micro-RNA variants we examined were altered in rats with heart failure compared to the healthy rats. Interval training changed 18 of these back towards healthy levels. Several of the relevant micromolecules are known to play a role in both calcium management and the electrical conduction system of the heart, but the most interesting thing is that we discovered new micro-RNAs that can play an important role in heart failure,” says Stølen.

This article has mostly considered the effects of high-intensity interval training. But the study also includes a group of rats that trained more sedately.

The rats in this group ran the same distance and thus did as much total training work as the rats in the interval training group. However, they had to exercise longer each time since they trained at a lower intensity. Stolen notes that this form of training also resulted in several health improvements.

But, he emphasizes, the vast majority of improvements were greater with interval training. “For example, we were able to induce cardiac fibrillation in five of eight rats after a period of moderate exercise, and their pumping capacity had only improved half as much as in the interval training group.”