Editor: This article is the first of a two-part series. Check out Part Two here.
If you have ever injured your muscles – either by exercising a tad bit too much or due to some unfortunate accident?
You probably know how important it is to have healthy, functional muscles for your day-to-day activities. The good news is our muscles are able to heal after a few days/weeks of rest. But what allows us to regain movement after minor muscle damage?
In short, an injury triggers a highly regulated sequence of events that is responsible for regenerating our skeletal muscles. These events would thereby allow us to go back to being our physically active selves.
Still skeptical about the importance of skeletal muscle?
Skeletal muscle accounts for ~40-45% of our body mass. From producing movement to generating body heat to regulating our core temperature, the skeletal muscle is a vital organ of the muscular system with a wide range of important functions. They act as reservoirs of carbohydrates and amino acids, thus playing a role in maintaining blood glucose levels.
Perhaps, one of the most interesting aspects of skeletal muscles is their ability to regenerate themselves after an injury. Just like every other process in our body, skeletal muscle regeneration is highly regulated. However, this ability depends on the type of injury – if it is too severe, the muscle will not be able to regenerate or may regenerate imperfectly, therefore resulting in poor muscle function. Age-related muscle frailty and muscle inflammation have been partly attributed to decreased regenerative ability.
In some genetic diseases, this regeneration process is impaired, which can result in severe muscle weakness, loss of muscle mass, inflammation, and pain. In severe cases, patients may have reduced life expectancy, become wheelchair-bound, or even develop cardiac and respiratory complications. In some cases, some patients may even experience poor eyesight or hearing loss.
Now that you’ve gotten a rough idea of how important the skeletal muscle regeneration process is, the aim of this article is to introduce you to the key player of skeletal muscle regeneration, satellite cells and the environment they are in, along with other cell types that are important for the process.
Stem cells - here we go again!
To understand the process of regeneration, we have to familiarise ourselves with the key player responsible, muscle stem cells.
So, what are all these stem cells that you keep hearing about in the media?
Stem cells are characterised by their ability to self-renew and differentiate into a particular cell type. Cellular differentiation refers to the process of cells becoming specialised/restricted to a particular cell type. Stem cells are classified according to their potency or developmental potential (See Figure 1). Potency simply refers to the ability of stem cells to develop into different cell types.
For example, fertilised egg (zygote) contains stem cells that can give rise to all types of cells. These cells are called totipotent stem cells, which gives rise to both the body and the placenta. As we grow and develop, stem cells begin to lose their potency, meaning they become limited in the different types of cells they can give rise to (See Figure 1). Totipotent cells lose potency to become pluripotent cells. Pluripotent cells lose potency to convert into cells that can give rise to only a certain lineage of cells. These types of stem cells are known as multipotent stem cells. A good example of this includes haematopoietic stem cells, which can only give rise to blood cells such as T-cells, B-cells, and macrophages. Multipotent stem cells further lose their potency to become unipotent stem cells, which can give rise to only one type of cells. For instance, muscle stem cells can only produce more muscle cells.
Figure 1: Classification of stem cells according to their developmental potential. The arrow from left to right represents the decrease in cell potency. Totipotent cells are found only in fertilised eggs (i.e. zygote). The difference between totipotent and pluripotent cells is that totipotent stem cells can give rise to tissue found outside the embryo, such as the placenta and umbilical cord. In adult humans, only multipotent and unipotent cells are present.
To simplify the concept of potency, think of stem cells as a mass of wet clay, potency as the loss of moisture, and fully differentiated cells as completely hardened pottery. The wet mass of clay can be moulded to form any shape! Let’s say you form a circle, the clay dries a bit (loss of moisture), so it can only be changed into shapes that are variations of a circle. The more it hardens, the more difficult it becomes to change its shape.
Introducing the rescuers - satellite cells!
Muscle stem cells are commonly referred to as satellite cells. This population of cells resides between the basal lamina and the myofiber plasma membrane (See Figure 2).
Figure 2: Schematic of the location of satellite cells in adult mammals (not to scale). Essentially, the basal lamina is an extracellular matrix, and it surrounds the myofiber within the muscle. As you can see in this diagram, you can find satellite cells residing between the basal lamina and myofiber plasma membrane.
Satellite cells in our bodies are unipotent. All the satellite cells present are heterogeneous (meaning nonidentical) in nature. Different subgroups are present which express different proteins, so are expected to vary in their roles. Upon activation of satellite cells, they proliferate rapidly to increase their numbers. The majority of the satellite cell population can divide to form daughter cells called myoblasts. These myoblasts differentiate into myocytes which subsequently fuse to form myotubes. At each stage, the satellite cells produce specific transcription factors that are critical for their transition/maturation to the next stage. Therefore, researchers typically use transcription factors as “markers” to determine which maturation stage the satellite cells are in. Different types of “markers” are also used to identify subpopulations of satellite cells.
Eventually, these myotubes can fuse together to form a single myofiber (or in other words, a muscle cell). Several myofibers can then bundle together to form a fascicle, while several fascicles can group together to form a muscle.
Figure 3: Schematic of how satellite cells (SC) differentiate into myotubes. Different colours in the SC population represent their heterogeneity (i.e. they are not identical). While the majority of satellite cells are activated to give rise to myoblast progeny, a small number are kept as reserves, which are only to be activated if there is severe muscle damage. After that, mono-nucleated myoblasts (which contain a single nucleus) differentiate to form myocytes which fuse together to form multinucleated (containing multiple nuclei) myotubes. A subset of the satellite cell population then undergoes cell-division to give rise to more satellite cells in order to restore the number of cells. Figure created on BioRender.
The essential helpers
We tend to think of cells as if they exist in isolation. Though in fact, cells are functioning in a three-dimensional environment within our bodies, where they are constantly interacting with surrounding cells such as neurons and blood cells. Occasionally, they are also interacting with molecules secreted by themselves.
In the case of stem cells that exist within a micro-environment that is made up of different cell types, signals from surrounding cells are called the stem cell niche (See Figure 4). It comes with no surprise that the behaviour and function of stem cells are under the control of the components of the niche.
Under healthy circumstances, the signals in the niche maintain the stem cells in a quiescent (dormant) environment. Upon injury, the subsequent activation, proliferation, and differentiation of satellite cells are regulated by different cell types both within and outside the niche. The important cell types involved in skeletal muscle regeneration are highlighted below.
Figure 4: A schematic overview of the satellite cell niche. Multinucleated myofiber is shown (at the bottom of the picture). Above it resides the satellite cell population. Surrounding both the muscle fibre and the satellite cell population in the extracellular matrix (ECM). The skeletal muscle's ECM has a distinct composition consisting of various macromolecules. The niche consists of resident macrophages and neutrophils along with fibroblasts; fibroblasts make up a small proportion of skeletal muscle but are functionally important. Other cell types are also present in lower numbers (not shown). Capillaries are present to supply oxygen to active muscle cells (at the top of the picture). Endothelial cells forming the inner lining of the blood vessel are referred to as the endothelium. Red blood cells and different types of white blood cells are also present in the blood. Figure created on BioRender.
The cells of the immune system - white blood cells
You might be more familiar with white blood cells in the context of fighting pathogens such as bacteria and viruses.. But, the involvement of white blood cells is also important for proper skeletal muscle regeneration. In the satellite cell niche, there are resident macrophages and neutrophils in the inactive state. Once they are activated, these white blood cells are able to recruit additional neutrophils and macrophages along with T cells and macrophages.
Fibroblasts
Surrounding the muscle fibres in the extracellular matrix (ECM) (See Figure 4). This is a layer of non-cellular components consisting of molecules such as collagen, laminin, and elastin. The ECM not only provides mechanical stability to the muscle fibres and satellite cell niche but also aids muscular contraction.
Most of the major components of ECM are produced by cells called fibroblasts. It has been shown that fibroblasts are crucial for skeletal muscle regeneration. Removal of fibroblasts in mice resulted in early differentiation of satellite cells and poor regeneration of skeletal muscle fibre.
Blood vessel and endothelial cells
Every cell within our body requires oxygen. To ensure a consistent oxygen supply, capillaries are in close proximity to muscle fibres.
Endothelial cells are a single layer of cells that form the inside of blood vessels. They control the passage of cells and molecules present in the blood to the target tissue. Moreover, endothelial cells produce a growth factor vascular endothelial growth factor (VEGF), that brings about the formation of new blood vessels (called angiogenesis).
Interestingly, VEGF levels in the blood increase after exercise. Skeletal muscle regeneration is aided by angiogenesis, and it has been shown differentiation of satellite cells is linked to angiogenesis, thereby highlighting the importance of adequate blood supply to the injured muscle.
Numerous signalling molecules are released by cells within the satellite cell niche upon skeletal muscle damage. All these signalling molecules have an effect on satellite cells, whereby they bring about their activation, proliferation, and differentiation in a timely regulated manner.
Even though a lot of signalling pathways involved in regulating satellite stem cell behaviour has been understood, the entire plethora of signalling molecules acting on satellite stem cells have not been fully characterised. Even molecules like nitric oxide have an effect on skeletal muscle regeneration. In fact, mice with an impaired nitric oxide signalling pathway, show defective skeletal muscle regeneration with myoblasts poorly differentiating.
Why you should care?
As mentioned previously, the numerous factors that control and regulate satellite cells are not known.
The satellite cell niche changes as we grow old and our regenerative ability decreases, by understanding the process of entire skeletal muscle regeneration process we can potentially tackle muscle problems associated with ageing, such as immobility, muscle weakness, and muscle pain.
Furthermore, this knowledge of skeletal muscle regeneration can be used to design therapies and drugs to tackle muscular dystrophies. For example, three dimensional scaffolds can be engineered to be placed in the severely injured muscle to replace muscle mass, for this the composition and role of ECM in satellite cell niche have to be elucidated. Since so much is still unknown, it is difficult to design specific drugs to tackle signalling molecules to improve muscle regeneration. Gene therapies and stem cell therapies are all being devised to tackle muscular dystrophies. The field of skeletal muscle is vast and exciting and consists of everything from bioengineering to designing gene therapies, and in need of novel ideas to aid characterisation of skeletal muscle regeneration and muscular dystrophies!
Now that you are familiarised with satellite cells and satellite cell niche, stay tuned for the next article to delve into the series of events that occur when skeletal muscle is injured, and for an insight into the world of muscular dystrophy and associated therapies currently in use!
Author: Israt Jahan, BSc Biotechnology with a Year in Research
Disclaimer: All figures created using BioRender are intended solely for educational purposes and not for profit.
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