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MODERN TECHNIQUES AGAINST MUSCLE ATROPHY

Introduction

Skeletal muscle is a dynamic tissue that adapts to different stimuli by changing its size. A balance between anabolic and catabolic systems maintains the muscle mass in physiological conditions. If the homeostatic balance is altered, one system can dominate the other, leading to muscle hypertrophy or atrophy. Muscle atrophy can occur in several pathological settings. It is characterized by a decrease in the size of myofiber and a reduction in muscle function. Further, we will look at three diseases and their treatments against muscle atrophy

Part I - Cancer Cachexia

Cancer cachexia is a hypercatabolic disease as a consequence of cancer. It is characterized by reduced food intake and abnormal metabolism, leading to muscle atrophy and sometimes also decrease in fat mass (Figure 1). It is estimated that about 50% of all human cancer patients will develop cachexia, and that an estimated 20% of the cancer patients dies from cachexia (Aoyagi et al, 2015). It has a drastic impact of a patient’s quality of life and is associated with poor responses to chemotherapy and decreased survival rates. Treatments focus on appetite stimulants, palliation of symptoms and reducing patient distress.

Figure 1 Showing how cancer can affect different parts of the body

What is it?

The most obvious symptom of cancer cachexia is loss of skeletal muscle. Myostatin and activin A are endogenous myokines, negative regulators of muscle growth, determining both muscle fiber number and size. During cancer cachexia the blood has higher concentrations of these substances than in healthy individuals. The exact mechanism behind this disease is still not entirely clear, but it appears that several factors interact with each other. Further, we will discuss the most relevant ones, as well as their treatment options.

Treatments that can increase muscle mass and physical performance may be a promising option. There are currently no pharmacological agents approved for prevention or treatment of cancer cachexia, but several drugs are in clinical development, including protein stimulating agents and drugs targeting inflammatory cytokines that promote skeletal muscle catabolism.

Inflammatory response

The competition for nutrients between the tumor and the host, as well as the inflammatory response lead to metabolic disorders that include a large nitrogen flow from the skeletal muscle to the liver. Pro-inflammatory cytokines are peptides that are important in cell signaling and is produced because of the systemic inflammation caused by cancer. They are a part of the host response. The body produces acute phase response (APR) proteins which involves the production of C-reactive protein (CRP) (Fearon et al, 1999). The concentration of CRP is an accurate measure of the amount of pro-inflammatory cytokines in the body. The APR proteins accelerates weight loss by increasing muscle catabolism and the transfer of amino acids from muscle anabolism towards APR protein synthesis.

Appetite stimulants

To avoid or reduce muscle atrophy, it is important that the body obtains adequate nutrition, and since many cancer patients struggle to keep their food intake up, appetite stimulants can be prescribed to cancer patients. Ghrelin is a gastric peptide hormone present in mammals. It is an orexigenic hormone which means that it stimulates the appetite (Cheung and Wu, 2013). It is involved in several physiological functions such as stimulation of growth hormone secretion, gastric acid secretion and motility. The ghrelin concentration is increased in individuals with cachexia, most likely as a compensatory mechanism.

Capromorelin and anamorelin are growth hormone secretagogues (GHS), which means that they stimulate the secretion of growth hormone (Zollers et al, 2016). GHS compounds mimic ghrelin, and they are therefore also called a ghrelin receptor agonist. In a clinical study completed on dogs with reduced appetite, Capromorelin showed to be an effective treatment for stimulation of appetite in dogs, where 68.6% of owners classified the treatment a success, compared to 44.6% of the placebo treated dogs. In two studies showing the effects of anamorelin on humans with cancer cachexia, treatment for 12 weeks had a favorable clinical response in patients (Garcia et al, 2014). The lean body mass increased in 86.3% of the anamorelin treated patients, against a mean lean body mass decrease in 94.7% of the placebo group.

β2-adrenergic agonists and antimyostatic combination therapy

β2-adrenergic receptors are transmembrane receptors that interact with epinephrine to stimulate adenylyl cyclase by activating the G-protein which leads to the dissociation of its alpha subunit charged with GTP. β2-adrenergic agonists are potent muscle growth promoters in many animal species resulting in skeletal muscle hypertrophy and reduction of the body fat content. Formoterol is a long-acting β2 agonist (LABA) that exerts a powerful action on the cardiac and skeletal muscle by activating the rate of protein synthesis and inhibiting the rate of muscle proteolysis. A recent study in cachexic mice by Toledo et al (2015) showed complete reversal of muscle atrophy in experimental cancer cachexia in mice by a combination of formoterol and the antimyostatic drug called activin type II receptor inhibitor (ActRIIB).

Figure 2 Shows survival of tumor-bearing mice, period of 40 days.

The graph (Figure 2) shows the survival of the tumor-bearing mice over a period of 40 days. The blue line shows the untreated mice. Each group had 8 individuals. The group treated with a combination of formoterol and ActRIIb displayed a significantly longer survival rate than the untreated mice. Circulating inflammatory cytokine concentrations (TNF-alpha, IL-1 and IL-6) were significantly lower in the groups receiving any of the two or a combination when compared to the untreated group. They also had a considerably higher hemoglobin count, hematocrit value and platelet count than the untreated group. The combined treatment also decreased the number of metastases. The results from this study show great prospects for an effective therapy in both animals and humans (Figure 3).

Figure 3 Combined treatment gives increased quality of life and higher survival rates

Future prospects

Since cancer cachexia mainly is due to a large number of cooperating factors, it is clear that multidimensional treatments represent the most useful approach for cachexia in advanced cancers. However, it is still currently regarded as a non-curable disease, and even though the palliative management of the disease has improved largely in the past decade, there is still no magic cure. Nevertheless, several interesting approaches are undergoing clinical trials.

Part II - Diabetes Type 2

Diabetes mellitus, commonly known as diabetes refers to a group of diseases where the body does not produce enough insulin, or does not use it efficiently (insulin resistance). Insulin is one of the main hormones produced by pancreatic beta cells, and it stimulates anabolic and storing processes in cells. It allows cells in the adipose tissue and muscles (containing GLUT4 insulin dependent transporters) to take up glucose and use it as a source of energy for proper function. There are several types of diabetes: gestational diabetes, diabetes LADA, diabetes MODY, double diabetes, type 3, secondary diabetes, steroid induced diabetes, type 1, type 2 etc. Research articles used in this essay is based on the latter one, which is the most common form of diabetes.

Importance of insulin

When the body lack insulin, plasma glucose level increases, and the intermediary metabolism is altered. In case of protein metabolism, insulin deficiency will increase protein catabolism, resulting in negative nitrogen balance as well as dehydration due to potassium loss. Regarding fat metabolism, mobilization of adipose deposits increases (lipemia) to cover energy needs since most of the cells are unable to uptake glucose. Oversupply with FFAs inhibit complete utilization of AcCoA, and ketone bodies appear in the plasma and urine causing sodium loss. Insulin deficiency also increase glycogenolysis, resulting in even higher levels of glucose. Reabsorbing capacity of renal tubules exhausts (reach level of 10 mM), and glycosuria follows. Energy source get lost, and osmotic diuresis occurs. Latter contribute to dehydration which can exacerbate the ketoacidosis. This is a serious medical emergency and can be lethal if not treated properly.

KLF15 and WWP1

Diabetes can evidently cause various health problems like macro- and microvascular diseases, neuropathy, kidney failure, retinal demage, gastroparesis, decline in muscle mass etc. The latter one is commonly referred to as muscle atrophy. The underlining mechanism for this phenomenon is uncertain. A research group led by Professor Wataru Ogawa at the Kobe University Graduate School of Medicine revealed that elevation of blood sugar levels leads to muscle atrophy and that two proteins, WWP1 and KLF15, play key roles in this phenomenon (Hirata et al, 2019). According to the study, elevated blood sugar level slows down degradation of transcriptional factor KLF15 (Krüppel-like factor 15). This protein is increased in diabetic mice, and mice lacking this protein (specifically in muscles) are resistant to diabetic muscle atrophy. WWP1 (ubiquitin ligase) binds ubiquitins to KLF15 improving degradation. Rising blood sugar level result in decreasing amount of WWP1, followed by increased KLF15. Development of a drug that either weakens the function of KLF15 or strengthens WWP1 could be a possible treatment.

Combination of alfacalcidol and exercise

The ability of alfacalcidol (ALF) and low-intensity aerobic exercise (Exe) to inhibit muscle atrophy have newly been examined (Akagawa et al, 2018). The purpose of the study was to clearify the effects of a combination therapy of these two. Twenty-week-old OLETF rats (diabetes type 2 model) were putted into different groups consisting of 8-10 rats. The groups were ALF (alfacalcidol orally 0.1 μg/kg/day), Exe (treadmill exercise at 10 m/min, 60 min/day, 5 days/week), Comb (ALF and Exe), and Cont (diabetes type 2 control). Blood glucose levels, cross-sectional area of tibialis anterior muscle fibers, and relative quantities of muscle anabolic markers (Pax7, MyoD, myogenin) and catabolic markers (Atrogin-1, MuRF1, REDD1) of the soleus muscle were measured after treatment for 2 or 6 weeks. They were compared to parameters of LETO rats (non-diabetes type 2) to see if OLETF rats (diabetes type 2 model) recovered to the same level.

Anabolic and catabolic markers are measured. Satellite cells are precursors to skeletal muscle cells, and Pax7 is important for their activation and normal function. Satellite cells differentiate into myoblasts, where MyoD plays an important role. Following myoblasts differentiate into myotubes controlled by myogenin. MuRF1 and Atrogin-1 (promoted by myostatin) is catabolic genes releated to several types of muscle atrophy. REDDI (stimulated by glucocorticoids) suppresses mTOR which is a kinase controlling many fundamental cell processes like protein synthesis. It controls cell growth in response to cellular energy, nutrients, stress and growth factors (Hall, 2008).

Results

After 2 weeks combination treatment increased MyoD expression and decreased MuRF1 expression. Individually, alfacalcidol or exercise remarkably decreased Atrogin-1 or MuRF1 expression. The cross sectional area is signficantly increased in comparison with the control group, as seen in Figure 4. Exercise therapy significantly increased the area compared with alfacalcidol treatment. Individually alfacalcidol and exercise could not recover the area to that of non-T2DM control rats (LETO). However, a combination significantly recovered the area to that of non-type 2 diabetic control rats (LETO group). After 6 weeks of treatment, both alfacalcidol and combination treatments decreased REDD1 and Atrogin-1. There were no significant difference in the anabolic markers. Alfacalcidol, exercise and combination therapy enchanted the area signficantly in comparison with control group. Anyhow, the CSAs in those groups were still significantly smaller than of non-T2DM rats (LETO group).

Figure 4 Cross sectional area after (A) 2-weeks group and (B) 6-weeks group.

Exe and ALF seperately represses Atrogin1 and MuRF1. Previous studies have demonstrated that activated vitamin D as well as exercise inhibit myostatin, and by that stimulate myogenic differentiation. Seperately, they did not increase expressions of anabolic genes. Just a combination of ALF and Exe could increase expression of MyoD and repress REDDI (decrease endogenous glucocorticoid level). These results indicate that a combination therapy is recommended for treatment of muscle atrophy in diabetes type 2.

Part III - Spinal Muscular Atrophy

SMA stands for Spinal Muscular Atrophy and is a neuromuscular disease. The disease can be divided into five different types. The different types occur at different stages of life, and have varying degrees of severity and symptoms. The following paragraphs focuses on SMA type1 (SMA1).

SMA1 occurs between 0-6 months of age, and without treatment, death normally occurs before two years of age (Eugenio et al, 2018). SMA1 is the number one cause of death in infants. Today there is no cure against SMA, but the knowledge on this field is increasing rapidly. There are some methods which can increase the lifespan, and decrease the symptoms of the patient (Gamze et al, 2017).

Cause of the disease

The cause of the disease is due to deficient of a vital protein called survival of motor neurons (also called SMN) in the spinal cords lower motor neurons. Lack of this protein leads motor neurons to die and the muscles that are controlled by the neurons to waste away (Mendell et al., 2017)

Figure 5 Healthy individuals and people with SMA.

Treatment

Until just a few years ago there was no real treatment for spinal muscular atrophy. There were only support of the patients to slow down, or avoid some symptoms of the condition. There is still no cure for SMA today, but there are newer and more efficient methods to treat spinal muscular atrophy. Two of the most promising methods are gene therapy and nusinersen. A common denominator for both methods is that the goal is to increase the amount of the SMN protein. (Vamshi et al, 2018).

Nusinersen

Besides the SMN1 gene, the body also produces the SMN2 gene which is different from SMN1 by one nucleotide. SMN2 is a less effective form of SMN1, and SMN2 mainly produce nonfunctional SMN protein. Nusinersen is able to increase the amount of functional SMN protein produced through the SMN2 gene. (Gamze et al, 2017). Because we know the sequence of the gene leading to SMA, we can synthesize nucleic acid which is then able to bind to mRNA produced by the gene which is causing SMA. Nusinersen will then regulate the function of the defect gene sequence.

Nusinersen will help to increase the production of SMN protein through the SMN2 gene instead of the SMN1 gene. Nusinersien binds a splicing silencer region on the pre-mRNA of the SMN2 gene just after exon 7. This prevents exon 7 from being skipped, and it will then be included in the mRNA, leading to a functional full-length version of the SMN protein (Vamshi et al, 2018). Nusinersen is not able to travel over the blood-brain barrier (formed by endothelial cells in a tight junction and works as a physical barrier, and is due to this injected directly into the spinal canal. (Abbott et al, 2006)

In the USA, nusinersen was approved as a drug in 2016, and in Europe in 2017. As nusinersen is such a new treatment, the knowledge is still not complete. However, two studies performed on both newborns and children with later onset SMA concludes that newborns treated with nusinersen had a bigger chance to have a longer life, and also had a better motor function. It is also important to start the treatment with nusinersen as early as possible as the treatment will have a better result (Gamze et al, 2017).

Gene Therapy

SMA is just affecting one gene, the SMN1 gene, and therefore gene therapy could be an optional treatment. When performing gene therapy, the goal is to either change the genetic material of the cells or to deliver a new and healthy gene into the cells. In SMA, gene therapy can be performed in two different forms to increase the level of SMN expressed:

1. By injecting into the veins, a virus vector containing the SMN1 gene to replace the current SMN1 gene in the body
2. By adjusting the SMN2 gene to increase the amount of functioning SMN protein (Gamze et al, 2017)

To get the SMN1 gene into the cells as described in the first method, a virus is used as a vector. The virus type used as vector is called adeno-associated virus (AAV9), and they are used due to their ability to cross the blood-brain barrier. In a study published in November 2017, children with SMA type 1 were given the AAV9 during one occasion. As a conclusion of the study, the treatment leads to a longer life, increased motor function compared to untreated SMA type 1 patients (Mendell et al, 2017).

Figure 6 Gene therapy approach for SMA with scAAV9 vector

Conclusion

There is still no cure against muscle atrophy in cancer cachexia, diabetes mellitius and SMA. However, there are treatments which can increase the life span and life quality. The knowledge in this field is increasing rapidly, and a lot of research is going on. Muscle atrophy in cancer cachexia treatment focus on appetite stimulants, palliation of symptoms and reducing the distress. In diabetes mellitus, there are no drugs on the market today for treatment of muscle atrophy. Nevertheless, a drug that weakens the function of KLF15 or strengthens WWP could eventually become a treatment. Additionally alfacalcidol combined with exercise could be an option as well. SMA treatment focuses on increasing the amount of the SMN protein. Hopefully, we will have enough knowledge in the near future to cure muscle atrophy in all of the three conditions.

References

Abbott, N. J.; Rönnbäck, L.; Hansson, E. (2006). Astrocyte–endothelial interactions at the blood–brain barrier. Nature Reviews Neuroscience 7: 41-53

Akagawa, M.; Miyakoshi, N.; Kasukawa, Y.; Ono, Y.; Yuasa, Y.; Nagahata, I.; Shimada, Y. (2018). Effects of activated vitamin D, alfacalcidol, and low-intensity aerobic exercise on osteopenia and muscle atrophy in type 2 diabetes mellitus model rats. Public Library of Science One. 13(10): e0204857.

Aoyagi, T.; Terracina, K. P.; Raza, A., Matsubara, H.; Takabe, K. (2015) Cancer cachexia, mechanism and treatment. World Journal of Gastrointestinal Oncology 7(4): 17–29

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Hirata, Y.; Nomura, K.; Senga, Y.; Okada, Y.; Kobayashi, K.; Okamoto, S.; Ogawa, W. (2019). Hyperglycemia induces skeletal muscle atrophy via a WWP1/KLF15 axis. JCI Insight Vol 4 (4): e124952

Mendell, J. R.; Al-Zaidy, S.; Shell, R.; Arnold, W. D.; Rodino-Klapac, L. R.; Prior, T. W.; Lowes, L.; Alfano, L.; Berry, K.; Church, K.; Kissel, J. T.; Nagendran, S.; L’Italien, J.; Sproule, D. M.; Wells, C.; Cardenas, J. A.; Heitzer, M. D.; Kaspar, A.; Corcoran, S.; Braun, L.; Likhite, S.; Miranda, C.; Meyer, K.; Foust, K. D.; Burghes, A. H. M.; Kaspar, B. K. (2017). Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy. The New England Journal of Medicine 377: 1713-1722

Mercuri, E.; Darras, B.T.; Chiriboga, C. A.; Day, J. W.; Campbell, C.; Connolly, A. M.; Iannaccone, S. T.; Kirschner, J.; Kuntz, N. L.; Saito, K.; Shieh, P. B.; Tulinius, M.; Mazzone, E. S.; Montes, J.; Bishop, K. M.; Yang, Q.; Foster, R.; Gheuens, S.; Bennett, F.; Farwell, W.; Schneider, E.; De Vivo, D. C.; Finkel, R. S. (2018) Nusinersen versus Sham Control in Later-Onset Spinal Muscular Atrophy. The New England Journal of Medicine 378: 625-635

Rao, V. K.; Kapp, D.; Schroth, M. (2018) Gene Therapy for Spinal Muscular Atrophy: An Emerging Treatment Option for a Devastating Disease. Journal of Managed Care & Specialty Pharmacy 12: 3-16

Toledo, M.; Busquets, S.; Penna, F.; Zhou, X.; Marmonti, E.; Betancourt, A.; Massa, D.; López-Soriano, F.; Han, H.; Argilés, J. (2015). Complete reversal of muscle wasting in experimental cancer cachexia: Additive effects of activin type II receptor inhibition and b-2 agonist. International Journal of Cancer 138 (8): 2021-2029

Zollers, B.; Wofford, J.; Heinen, E.; Huebner, M.; Rhodes, L. (2016). A Prospective, Randomized, Masked, Placebo‐Controlled Clinical Study of Capromorelin in Dogs with Reduced Appetite. Journal of Veterinary Internal Medicine, vol 30 (6): 1851-1857

Additional Materials

Wikipedia. Antisense therapy. Retrieved April, 2019 from https://en.wikipedia.org/wiki/Antisense_therapy

Diabetes Community UK. Diabetes Types. Retrived April, 2019 from https://www.diabetes.co.uk/diabetes-types.html

National Institute of Diabetes and Digestive and Kidney Diseases. (November, 2016) What is diabetes? Retrieved from https://www.niddk.nih.gov/health-information/diabetes/overview/what-is-diabetes

UVMB, Department of Physiology and Biochemistry. Endocrinology [PowerPoint presentation] Retrieved April, 2019

Figure 1-6: Made by the essay group