Duchenne Muscular Dystrophy: Forward for Casual Readers

Duchenne Muscular Dystrophy: Forward for Casual Readers

            Duchenne Muscular Dystrophy (DMD) is a genetic disorder of the muscles that causes severe muscle wasting and leads to death at a young age. DMD affects about 1 in every 3500 male births and is much rarer in females. Afflicted are born without symptoms, but muscle weakness first appears around the ages of 3 to 5. Initially, it affects only the distal muscles of the legs and arms, but it rapidly progresses to all muscles in the body. By the age of 12, most patients lose the ability to walk and are wheelchair bound. Death usually occurs before the age of 30 due to weakening of the heart and lungs.

            DMD is caused by a mutation of the DMD gene, which codes for the protein dystrophin.1 It is an inherited X-linked recessive disorder, meaning the mutation causing the disease is a recessive trait located on the X chromosome. The X chromosome is one of two sex-linked chromosomes, the other being the Y chromosome. This is the reason why the disease is so much more common in males; females have the sex chromosomes XX, and so if one of their X chromosomes has the recessive mutation, the other will still work properly (this is known as a female carrier). Thus, they need two mutated X chromosomes to express the disease. Males, who have the sex chromosomes XY, only have one X chromosome. So if that chromosome carries the mutation, there is no other functional one to use.

            About two thirds of DMD patients acquire the disease from inheritance, and the remaining third from spontaneous new mutations. DMD is not caused by the same mutation in everyone. A variety of different mutations can be responsible. What these mutations all have in common is that they are frameshift mutations.2 Genes are expressed by transcribing DNA into mRNA, and then translating the mRNA into proteins made of a chain of amino acids. In this process, DNA is read three nucleotides at a time, corresponding to one amino acid per three bases. Frameshifting mutations are any mutations that change the nucleotide sequence such that this reading frame is thrown off. For example, deleting or inserting a nucleotide at any given spot will change the three-nucleotide code at that spot, while shifting all subsequent nucleotides forward or backward, completely changing the makeup of each three-nucleotide unit and therefore the amino acid that corresponds to that unit. The result is a protein completely unrecognizable from its normal form.

            In DMD patients, this ultimately results in a complete loss of functional dystrophin. Dystrophin is a protein found inside of muscle cells attached to the sarcolemma (muscle cell membrane). Its role is to connect the cytoskeleton, or the interior structural framework of the cell, to the extracellular matrix, a network of structures and molecules outside of and between cells. It does this by binding to a complex of proteins bound to the cell membrane, which themselves are bound to proteins of the extracellular matrix.3 This provides support and stability to the sarcolemma, and helps to orient other important membrane proteins.

            Without dystrophin, the sarcolemma becomes much more fragile, and is prone to tearing. This allows calcium ions from outside the cell to flow into the cell at excessive levels. Calcium ions are used in healthy muscle cells in the process of muscle contraction. At abnormally high levels however, calcium ions serve as activators of multiple processes inside the cell that cause inflammation and cell death.4 The high rates of cell death in DMD patients cause necrosis of muscles, which eventually cannot keep up with regeneration and begin to degrade. Muscle tissue is replaced with fibrous connective tissue and fat that does not contract, which contributes to the muscle weakness/degradation of DMD patients.

            DMD currently has no cure and is universally fatal. Treatment is limited to alleviating symptoms in order to improve patient quality of life and provide small delays in the progression of the disease. However, a number of promising genetic treatments are currently being studied. One such technique, called “exon skipping”, involves removing exons (large sections of DNA that constitute a subunit of the coded protein) that contain a mutation. Removing damaged exons keeps the reading frame intact, allowing for the expression of a shorter, but still functional dystrophin protein. Very recent research is also exploring the possibility of CRISPR/Cas9, a relatively new technique that allows for precise editing of the genome. If successful, such techniques could revolutionize the ability of doctors to prolong the lives of DMD patients in the coming years.

To read further, view these in-depth pages about the history, characterization, pathology, and treatment of the disease.

References

  1. Hoffman EP, Brown RH Jr. Kunkel LM. 1987. Dystrophin: the protein product of the duchenne muscular dystrophy locus. Cell. 51(6):919-28. DOI: 10.1016/0092-8674(87)90579-4
  2. Monaco et al. 1988. An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics. 2(1):90-95. DOI: 10.1016/0888-7543(88)90113-9
  3. Ervasti JM and Campbell KP. 1991. Membrane organization of the dystrophin-glycoprotein complex. Cell. 66(6):1121-1131. DOI: 10.1016/0092-8674(91)90035-W
  4. Burr AR and Molkentin JD. 2015 Genetic evidence in the mouse solidifies the calcium hypothesis of myofiber death in muscular dystrophy. Nature 22(9):1402-1412.

Reflection 3: Disease Themes

Duchenne Muscular Dystrophy (DMD) is a severe muscular disorder caused by mutation in the DMD gene, resulting in acute muscle wasting and weakness caused by the almost complete absence of functional dystrophin protein . Dystrophin itself plays a key role in the healthy function of muscle, providing support and strength to muscle fibers. As such, DMD is an extremely fatal condition, with virtually all cases leading to an early death before the age of 30 or 40. DMD and the muscular dystrophy family of diseases have been known of for centuries, but much of the knowledge surrounding it has been uncovered only relatively recently. In compiling the extensive literature covering this disease, the following themes were apparent to me.

Theme 1: Identification and Distinction – Genetic, Clinical, and Historical

            The evolution of the clinical and genetic understanding of DMD over time is truly fascinating. DMD was noticed for the very first time in the nineteenth century. It was named posthumously after Dr. Duchenne de Boulogne of France (1806-1875), who worked with and extensively documented muscular dystrophy patients. This was and is slightly controversial apparently, as muscular dystrophy was first identified decades before Duchenne supposedly first published about it, and it is disputed whether he or another contemporary physician contributed more. Not much more is known about DMD or related diseases for the better part of a century until the 1970, 80s and 90s. In these three decades understanding progressed rapidly; the gene responsible was identified and the protein encoded by that gene, dystrophin, was characterized (along with its critically important healthy state function, associated proteins, and disease state dysfunction). In the year 2000, many of the mutations responsible for DMD was characterized on the dystrophin protein itself by Norwood et al.1 This new knowledge has also allowed scientists to specifically characterize and distinguish others in the disease family such as Becker Muscular Dystrophy and Limb Girdle Muscular Dystrophy.

Theme 2: Biomolecular and Cellular Pathology – Dystrophin and the DAPC

            Doctors and researchers had a relatively poor understanding of the underlying causes of Duchenne’s and other types of muscular dystrophy for most of the period since its discovery. Between the 1860s and 1980s, just about the only new discovery about the pathology of DMD was that it was X-linked recessive in the 1950s.2 The understanding of what goes wrong, and how, at the cellular and biochemical levels has increased vastly since dystrophin was first discovered in 1987, however.3 A series of papers by Ervasti et al. in the early to mid-1990s characterized the transmembrane dystrophin-associated protein complex (DAPC).4 In the absence of dystrophin, this complex was found to be partly or wholly missing as well. In 1993, Petrof et al. found further support that dystrophin and the DAPC play a role in sarcolemma stability and strength, demonstrating that dystrophin deficient muscles were more prone to sarcolemma tearing.5 Thus, the loss of dystrophin results in the weakening the sarcolemma and ultimately triggers a number of degenerative pathways such as fibrosis and diminished muscular regeneration.

Theme 3: Treatment – Dystrophin Restoration and Gene Therapy

            There are currently no treatments that effectively cure DMD. As such, treatment options primarily consist of symptom management, delaying disease progression and improving quality of life for as long as possible. However, advances in biomedical technology in recent decades have shown very promising results in rescuing dystrophin expression at a genetic level. Current biochemical research is focused largely on two methods: exon-skipping and gene therapy. DMD normally arises from a frameshift mutation that has a highly disruptive effect on the synthesis of the entire protein. Exon skipping is one strategy to get around this, by inhibiting certain exons and excluding them from splicing, thus “skipping” them.6 Skipping the damaged exon while remaining in frame results in a truncated, but still partially functional dystrophin protein that will usually not manifest symptoms as severe as seen in DMD. Additionally, researchers are beginning to explore potential gene therapies able to restore proper expression of dystrophin by correcting mutant gene expression. Research here has sought to use adenoviruses as vectors of gene replacement. More recently, researchers have also begun looking into the use of Crispr/Cas9 gene manipulation, a relatively new and extremely precise method of genomic editing that may eventually have the potential to cut out damaged sections of DNA, and replace it with the correct sequence.

1. Norwood, F.L., Sutherland-Smith, A.J., Keep, N.H., Kendrick-Jones, J. 2000. The Structure of the N-Terminal Actin-Binding Domain of Human Dystrophin and How Mutations in This Domain May Cause Duchenne or Becker Muscular Dystrophy.  Structure 8:481

2. Walton JN. 1955. On the inheritance of muscular dystrophy. Ann Hum Genet. 20(1):1-13.

3. Zhang Y et al. 2020. Enhanced CRISPR-Cas9 correction of Duchenne muscular dystrophy in mice by a self-complementary AAV delivery system. Sci Adv. 6(8): eaay6812.

4. Ervasti JM, Campbell KP. 1991. Membrane organization of the dystrophin-glycoprotein complex. Cell. 66:1121-1131.

5. Petrof et al. 1993. Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proc Natl Acad Sci U S A. 90(8): 3710-3714.

6. Mann CJ, Honeyman K, Cheng AJ, et al. 2001. Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse. Proc Natl Acad Sci U S A. 98(1):42–47

Using the Proteasome to Treat Heart Failure: Protective Effect of PDE1 Inhibition

Using the Proteasome to Treat Heart Failure: Protective Effect of PDE1 Inhibition

Original Paper – PDE1 inhibition facilitates proteasomal degradation of misfolded proteins and protects against cardiac proteinopathy: https://advances.sciencemag.org/content/5/5/eaaw5870

Heart disease is the leading cause of death in the United States, and has been for some time. A significant number of these deaths involve heart failure (HF), a clinical condition in which the heart is unable to pump enough blood to keep up with the body’s needs. Heart failure may be categorized into two types according to the heart’s ejection fraction (the percentage of blood out of the total volume of blood in the left ventricle that is pumped out with each contraction): heart failure with reduced ejection fraction (HFrEF), characterized by an ejection fraction of less than 40%, and heart failure with preserved ejection fraction (HFpEF), characterized by normal ejection fraction (50%-70%; 41%-49% considered borderline), with an unhealthy reduction in overall blood volume in the left ventricle. In the United States, about one half of heart failure cases are HFpEF, though the ratio of HFpEF to HFrEF cases has begun to increase, and the former is soon expected to account for the majority of cases. Although significant advancements in treatment for HFrEF have lowered mortality in recent decades, there is still no effective pharmacological treatment for HFpEF, representing a significant unmet medical need.1

            Cardiac proteinopathy is a general category of diseases caused by increased misfolded proteins, and as a result increased proteotoxic stress (IPTS), in heart muscle cells. It is the job of the proteasome to degrade these misfolded proteins to maintain proteostasis and prevent proteotoxic stress. Targeted degradation by the proteasome occurs after the misfolded protein is bound by ubiquitin by a series of co-enzymes. Multiple ubiquitin units are subsequently added, and the 26S proteasome detects and degrades the poly-ubiquitinated protein, preventing them from aggregating. Past studies have implicated proteasome functional insufficiency in cardiomyopathy and progression to heart failure, highlighting proteasomal regulation as a potential target for treatment.2,4

            A previous study by contributor Alfred Goldberg, it was shown that post-translational modification of the 26S proteasome acts as a means of regulation, specifically through the phosphorylation of various subunits, some of which stimulated increased proteasomal activity. Misfolded protein aggregates act as inhibitors to proteasomal activity, making this a promising find in relation to proteostasis maintenance.5 In a later study, Goldberg et al. specifically identified a pathway for proteasome activation by cAMP, via the phosphorylation of the Rpn6 subunit by Protein Kinase A (PKA).6 Author Xuejun Wang identified a similar pathway for proteasomal stimulation by cGMP/PKG.7 These protein kinases are dependent on their respective cyclic nucleotides to function.

            Phosphodiesterase (PDE) is a family of enzymes that break phosphodiester bonds. It is the function of a specific cyclic nucleotide PDE, PDE1, to convert cAMP and cGMP to AMP and GMP respectively, thereby playing an important role in signaling regulation. By breaking down the cyclic nucleotides, PDE1 also effectively inhibits PKA and PKG, and thus their ability to stimulate proteasome activity. Knight and Yan identified PDE1 inhibition as a potential therapy for heart diseases.8 In this current study, the authors demonstrate the effectiveness of PDE1 inhibition in treating HFpEF in mice by upregulating proteasomal activity via increased activation of PKA and PKG.

             In this study, the authors demonstrate that PDE1 inhibition exhibits a protective effect against cardiac proteinopathy and IPTS, and show that this is done via enhanced proteasome activity in a PKG and PKA dependent manner. The experiments utilized a transgenic mouse model of cardiac proteinopathy, induced by the expression of CryABR120G (mutant CryAB), a misfolded mutant form of the protein CryAB known to cause human disease.1

The authors first show that PDE1 protein levels were significantly increased in mutant CryAB mice compared to wild type (healthy) CryAB mice and non-transgenic (NTG; no CryAB) mice. They also demonstrate the ability of PDE1 inhibition to stimulate the ubiquitin-proteasome system, using a green fluorescent protein, GFPdgn. Mice treated with IC86430, a PDE1 inhibitor, showed 40% lower GFPdgn levels than control mice. GFPdgn mRNA levels remained comparable between both groups however, suggesting that the reduced protein levels seen in experimental mice is due to post-transcriptional action.

            Following this, the authors proceed to show the therapeutic effects of PDE1 inhibition on HFpEF. In this experiment, 134 mutant CryAB mice were randomly assigned to two groups, one receiving PDE1 inhibition via IC86430, and the other receiving unaltered vehicle as a control. As expected of mutant CryAB mice, both groups displayed signs of HFpEF prior to treatment. Following the treatment period however, heart malfunction in PDE1-inhibited mice was significantly reversed, with some functions returning to normal expected NTG levels, while the control group did not show the same improvement. Notably, during the 4-week treatment period as well as the 6 weeks following the end of treatment, none of the PDE1-inhibited mice died, while during that same period, 50% of control group mice did die. These results support the ability of PDE1 inhibition to treat cardiac dysfunction, and also potentially implicates the protective effect of PDE1 inhibition as a long term one.

            Experiments were also performed to support the mechanism of the therapeutic effect of PDE1 inhibition, that being the stimulation of proteasome activity by PKG and PKA, following their activation by cGMP and cAMP. After showing that PDE1 inhibition stimulates proteasomal degradation of GFPdgn, a general surrogate substrate of the proteasome, the authors wanted to show that the proteasome would target the disease-causing misfolded mutant CryAB specifically, which they confirmed utilizing a novel protein assay that can distinguish proteasomal degradation of misfolded proteins from other proteins undergoing general turnover. The mutant CryAB mice expectedly showed significantly higher levels of mutant CryAB protein, while NTG mice showed none. PDE1 inhibition significantly lowered mutant CryAB protein levels in mutant mice. Authors also found in this experiment a significant increase in Ser14 phosphorylated Rpn6 subunits in PDE1 inhibited mice, which previous studies have confirmed to occur through PKA. These findings support the proposition that PDE1 inhibition leads to increased levels of PKA, which in turn leads to increased phosphorylation of Rpn6 and thus increased proteasome activity, degrading misfolded proteins in the heart. Finally, the authors demonstrate that this process is dependent on PKA and PKG. Using another green fluorescent protein, GFPu, as the proteasomal substrate, the authors treated mice with a PKG inhibitor (KT5823) and a PKA inhibitor (H89) in addition to the PDE1 inhibitor. Inhibiting PKG and PKA significantly reduced the protective effect of PDE1 inhibition, indicating that this pathway is mediated by PKG and PKA.

            The findings of this paper uncover a new, potentially highly effective and longlasting treatment for heart failure, and in particular, HFpEF, a category of heart failure for which there are currently few therapeutic options. Future research can further explore the ability of PDE1 inhibition to treat cardiac proteinopathy by varying the onset and length of treatment; the beginning of treatment in this study was at the onset of the disease, and the authors predict an even greater effect if treatment is started earlier or longer lasting. Because this study only uses IC86430, similar studies might also be held with other PDE1 inhibitors to determine which might display the most efficacy. This study relates to our course in highlighting the extreme importance of the ubiquitin-proteasome system in preserving normal cell function, as well as the importance of protein quality control in disease prevention.

1. H Zhang, B Pan, P Wu, N Parajuli, MD Rekhter, AL Goldberg, X Wang. PDE1 inhibition facilitates proteasomal degradation of misfolded proteins and protects against cardiac proteinopathy. Sci. Adv. 5(5), eaaw5870 (2019). doi: 10.1126/sciadv.aaw5870

2. X Wang, J Robbins. Proteasomal and lysosomal protein degradation and heart disease. J. Mol. Cell. Cardiol. 71, 16–24 (2014). doi: 10.1016/j.yjmcc.2013.11.006

3. E Arbustini, P Morbini, M Grasso, R Fasani, L Verga, O Bellini, B Dal Bello, C Campana, G Piccolo, O Febo, C Opasich, A Gavazzi, VJ Ferrans. Restrictive cardiomyopathy, atrioventricular block, and mild to subclinical myopathy in patients with desmin-immunoreactive material deposits. J. Am. Coll. Cardiol. 31, 645–653 (1998). doi: 10.1016/S0735-1097(98)00026-6

4. J Li, KM Horak, H Su, A Sanbe, J Robbins, X Wang. Enhancement of proteasomal function protects against cardiac proteinopathy and ischemia/reperfusion injury in mice. J. Clin. Invest. 121, 3689–3700 (2011). doi: 10.1172/JCI45709

5. JJS VerPlank, AL Goldberg. Regulating protein breakdown through proteasome phosphorylation. Biochem. J. 474, 3355–3371 (2017). doi: 10.1042/BCJ20160809

6. JJS VerPlank, S Lokireddy, J Zhao, AL Goldberg. 26S Proteasomes are rapidly activated by diverse hormones and physiological states that raise cAMP and cause Rpn6 phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 116, 4228–4237 (2019). doi: https://doi.org/10.1073/pnas.1809254116

7. MJ Ranek, EJM Terpstra, J Li, DA Kass, X Wang. Protein kinase G positively regulates proteasome-mediated degradation of misfolded proteins. Circulation 128, 365–376 (2013). doi: https://doi.org/10.1161/CIRCULATIONAHA.113.001971

8. W Knight, C Yan. Therapeutic potential of PDE modulation in treating heart disease. Future Med. Chem. 5, 1607–1620 (2013). doi:10.4155/fmc.13.127.

Why I Study Biochemistry

Biochemistry is a highly comprehensive field of science with many reasons to study it, having importance in countless aspects of human life today. It has applications in medicine, from general practice to pharmaceutical and medical research, nutrition and food science, and environmental science, among others. Each of these areas is highly impactful to human health and wellness, and each of these require biochemical insight to determine how a number of their processes work, and what actions or procedures must be used to work in these areas.

Recent biochemical advances have been responsible for a number of important technologies we often take advantage of that serve critical functions in society today. Treatments for cancer and genetic disorders owe their success in large part to biochemical study of important underlying genetic structures and processes, such as the identification of the p53 gene as an important tumor suppressor. Drugs and pharmaceuticals are developed through the study of their effects on the many biochemical processes that run the body. The further study of biochemistry itself, along with many other disciplines within biology, is possible due to biochemical advances in detecting and purifying biomolecules. For example, Polymerase Chain Reactions (PCR), is a technique that is critical to biological study today for providing a method for easily and efficiently replicating a DNA sequence of interest to an observable quantity using a polymerase from T. aquaticus. Not only did this breakthrough simplify the process by which researchers could obtain DNA of interest for study, but it also allowed for the development of DNA testing services, for purposes such as criminal investigation or paternity/maternity testing that had previously been impossible due to limits in obtaining a testable quantity of subject DNA.

I chose to study biochemistry chiefly because I thought it to be the best overall starting point for my future career. As a pre-med student, at a college without a formal pre-med degree, biochemistry stuck out as the major with the broadest, as well as the most relevant, coverage of medicine-related science. In addition to coverage of biochemistry as a distinct discipline, the major also offers significant coverage of both biology and chemistry separately through its intro courses, and a variety of elective choices. The major thus covers many topics that medical schools expect their applicants to be versed in, including biochemistry, organic chemistry, general chemistry, calculus, and various branches of biology. This wide range of material is not only extremely helpful in preparation for medical school, it also provides the academic basis for a plethora of other scientific career paths should I change my mind about medical school for any reason, such as pharmacy school, doctorate programs, or even distantly related fields in public health or science administration. Its cross-disciplinary course distribution provides exposure to a wide array of subjects, which in turn allows for a diverse and wide-ranging education not found to quite the same extent in other studies.

Furthermore, the study of biochemistry in and of itself is the study of the systems that serve as the basis for life itself. Studying biochemistry allows one to study the structure and function of the innumerable biomolecules that make up life in a fundamental way, from basic amino acids and monomeric sugars, to complex multi part proteins and polysaccharides. If nothing else, it is at least a fascinating field of study providing a deep scientific insight to the many processes and systems instrumental for life. It serves as the bridge between chemistry, which provides understanding into the workings of nature, and biology, which provides an understanding of the components of life, using chemistry to explain how and why biological systems work.