The role of obscurin in muscle and why the R4344Q variant is unlikely disease causing

Muscle cells are a particularly fascinating type of cells from which all muscles in our bodies are composed of. Although the details of the molecular architecture differs between muscle types (e.g. between the heart and skeletal muscles such as the biceps), the basic mechanisms of contraction and relaxation are the same in every muscle:

First, an electrical signal from nerve endings in skeletal muscle or from neighbouring muscle cells in the heart triggers the release of calcium from an internal storage (the sarcoplasmic reticulum; subsequently abbreviated by “SR”). Calcium then activates the molecular ‘machine’ responsible for muscle contraction (the sarcomere). Finally, calcium is taken up into the SR again to end the contraction and allow the muscle to relax until the next electrical signal triggers another round of contraction.

The robust and precise functioning of these mechanisms is crucial for the health of our muscles and heart. In fact, genetic mutations affecting the genes and proteins that control these mechanisms often lead to severe diseases such as muscular dystrophy, heart failure or arrhythmias.

Obscurin is a giant protein located at the interface between the sarcomere and the SR. As the name (derived from the adjective ‘obscure’) suggests, the protein is very hard to study and we know still very little about what it does. What we do know is that it’s a very modular protein with segments that indicate structural functions and segments which suggest biological signaling functions. It has also been discovered that obscurin binds to many other muscle proteins including titin, myomesin, small ankyrins and others.

Simplified scheme of the molecular architecture of heart muscle cells and the localiation of phospholamban and obscurin.

Several early studies on muscle cells cultured on Petri dishes and studies on zebrafish reported that reducing the number of obscurin molecules or deleting parts of obscurin can lead to mishaped muscle fibrils and interferes with sarcomere organisation.1,2 In contrast, however, a later study reported that deleting obscurin in mice had no effect on the organisation of muscle fibrils and sarcomeres, but resulted in altered SR structure.3 Moreover, mice without obscurin were generally healthy, although somewhat more susceptible to muscle damage resulting from exercise.4 This indicates that obscurin is largely dispensable for normal muscle development and heart function in mice but could play a role in the adapting the muscle to mechanical stress. Further evidence for this hypothesis comes from the observation that obscurin seems to be enriched in cellular regions of muscle damage after exercise and from the observation that some regions of obscurin show strongly altered phosphorylation* patterns after intensive muscle exercise.5,6

While it’s still largely unknown what obscurin does exactly and how it does whatever it does, mutations in the gene that encodes the obscurin protein are increasingly being associated with  diseases such as heart failure (see 7 for a review). The first mutation associated with heart disease is the variant R4344Q** which was discovered by Arimura et al. in a patient with hypertrophic cardiomyopathy (a kind of pathological growth of the heart muscle).8 About ten years later, the lab of Prof. Aikaterini Kontrogianni-Konstantopolous at the University of Maryland (Baltimore) developed an R4344Q mouse model to further study whether this variant is harmful and found that these mice developed severe arrhythmias and that their hearts showed signs of dilated cardiomyopathy when subjected to cardiac stress.9 The authors of this study concluded that “(…) it is highly likely that the presence of the R4344Q mutation may render the carriers more susceptible to develop [cardiomyopathies]” (p.10).

Astonishingly, approximately 15% of black African Americans (about 1 out of 7) carry this mutation.10,7 Given the effects of this mutation observed in mice (dilated cardiomyopathy, fibrosis, arrhythmias), this would imply a significant disease risk for an alarming number of people. Indeed, as African Americans are statistically more often affected by heart disease, Hu et al. speculated that the R4344Q mutation is an important factor in this. If the R4344Q mutation really is a disease causing factor, one would expect that it has a major influence on the molecular properties of obscurin such as the strength with which it binds to other proteins or on the stability of the protein. In fact, Arimura et al. reported that the R4344Q reduces the binding to the sarcomeric protein titin, whereas Hu et al. found a new interaction between obscurin and a SR protein called phospholamban which they estimated to be strengthed by a factor of approximately 10.

However, due to the harmful effects observed in R4344Q mice and due to the fact that both titin and phospholamban are very important proteins in heart muscle cells, we were surprised that not even more African Americans are affected by heart disease. To put it another way: if the impact of the R4344Q mutation is as large as reported by these studies, why do not more carriers of this mutation develop severe heart disease? Is it possible that the R4344Q mutation is not harmful after all?

To answer this question, we set out to systematically investigate the impact of the R4344Q mutation on all interactions with other proteins known to take place in the affected region of obscurin. A major limitation of previous studies on the R4344Q mutation is that the interaction of obscurin with other proteins was assessed by methods that only allow a qualitative comparison at best. We, therefore, used methods which allowed us to directly measure the strength of an interaction between two proteins. Since we know from other muscle proteins that some genetic mutations cause disease by destabilising the protein, we also measured the stability of the affected obscurin region when exposed to increasing heat.

What we found was quite surprising to us!

First, we discovered that the binding of obscurin to phospholamban relies on the transmembrane region of phospholamban. In living muscle cells, this transmembrane region of phospholamban is inserted into the lipid membrane of the SR. This is surprising because such transmembrane regions are typically not accessible to other proteins unless they also have transmembrane elements. As far as we know, however, obscurin has no such transmembrane regions. We therefore tested whether obscurin and phospholamban interact in more physiological context. Using actual human cells, we didn’t detect any signs of such interaction. We therefore think that the interaction between obscurin and phospholamban is actually an in vitro artefact.

Second, we also quantified whether the R4344Q mutation influences a well established interaction between obscurin and the muscle protein titin. We did not find that the R4344Q mutation weakens this interaction at all. Although this contradicts Arimura et al., we used a more precise method and furthermore confirmed our result with second method that relies on a different measurement principle (isothermal titration calorimetry).

Finally, we also characterised the impact of the R4344Q mutation on protein stability. Although we found a very small, statistically significant decrease in stability, the difference is so small that it’s highly unlikely to be biologically relevant. We know from other mutations in structurally similar proteins, that mutations which are known to cause disease via protein destabilisation typically have a more than 8x larger effect as observed for the R4344Q mutation.

In summary, we found that the R4344Q mutation does not alter any known biological property of obscurin.

What does this mean for people carrying the R4344Q mutation?

At the very least it means that the R4344Q mutation is unlikely to cause harm in any of the ways previously proposed. Moreover, it further supports a strong argument that the mutation is not causing heart disease in humans at all and that it’s more appropriate to call it a benign variant rather than mutation (a term which most people associate with something bad). If you consider the age distribution of R4344Q carriers in a genetic sequencing database such as gnomAD (, you will find dozens of homozygous carriers***, many of which are 60 years or older. This clearly indicates that the R4344Q variant does not pose a high risk for serious heart disease. In agreement with this, the authors from a study published 2016 in the New England Journal of Medicine classified the R4344Q variant as “unambigously benign” – i.e. not disease causing.10

Does this exclude every logical possibility that the R4344Q variant can somehow increase your risk for heart disease? Probably not. However, everything we know so far indicates that the risk will be minor at most. If one really wants to figure out whether the R4344Q variant can increase the risk for heart disease, a clinical study of sufficient statistical power will be ultimately necessary. (Detecting small effects requires many participants.)

As discussed in our paper and elsewhere, many others factors, including socioeconomic disadvantages, contribute to the increased risk for cardiovascular and other diseases among African Americans.11,12 As genetic misdiagnoses risk causing more harm to patients and their families, it is paramount that evaluations of genetic variants are based on clear and sufficient clinical evidence.13 For people with the R4344Q variant who want to prevent heart disease, I think the best advice is to try and follow general guidelines for living a healthy life as e.g. outlined by the American Heart Association14 rather than to be concerned about the R4344Q variant.****


*Phosphorylation is a type of physiological chemical modification of proteins which can activate, inactivate or change the function of a protein in other ways.

**This notation indicates that the mutation leads to an exchange of the amino acid arginine (R) at position 4344 to glutamine (Q).

*** Since we humans have two copies of each chromosome, we also have two copies of each gene. If one of your genes has a specific genetic variation, you’re a heterozygous carrier of this variant. If both copies have the same variation, you’re a homozygous carrier.

**** That is if you have no other harmful mutations of course! Having actual symptoms of heart disease or family history of heart disease could be a warning sign that requires you to be seen by a doctor!


1. Kontrogianni-Konstantopoulos A, Catino DH, Strong JC, Sutter S, Borisov AB, Pumplin DW, Russell MW, Bloch RJ. Obscurin modulates the assembly and organization of sarcomeres and the sarcoplasmic reticulum. The FASEB Journal. 2006;20:2102–2111.

2. Raeker MÖ, Bieniek AN, Ryan AS, Tsai H-J, Zahn KM, Russell MW. Targeted deletion of the zebrafish obscurin A RhoGEF domain affects heart, skeletal muscle and brain development. Developmental Biology. 2010;337:432–443.

3. Lange S, Ouyang K, Meyer G, Cui L, Cheng H, Lieber RL, Chen J. Obscurin determines the architecture of the longitudinal sarcoplasmic reticulum. J Cell Sci. 2009;122:2640.

4. Randazzo D, Blaauw B, Paolini C, Pierantozzi E, Spinozzi S, Lange S, Chen J, Protasi F, Reggiani C, Sorrentino V. Exercise-induced alterations and loss of sarcomeric M-line organization in the diaphragm muscle of obscurin knockout mice. American Journal of Physiology – Cell Physiology. 2017;312:C16–C28.

5. Carlsson L, Yu J-G, Thornell L-E. New aspects of obscurin in human striated muscles. Histochemistry and Cell Biology. 2008;130:91–103.

6. Potts GK, McNally RM, Blanco R, You J-S, Hebert AS, Westphall MS, Coon JJ, Hornberger TA. A map of the phosphoproteomic alterations that occur after a bout of maximal-intensity contractions. J Physiol. 2017;595:5209–5226.

7. Marston S. Obscurin variants and inherited cardiomyopathies. Biophysical Reviews. 2017;9:239–243.

8. Arimura T, Matsumoto Y, Okazaki O, Hayashi T, Takahashi M, Inagaki N, Hinohara K, Ashizawa N, Yano K, Kimura A. Structural analysis of obscurin gene in hypertrophic cardiomyopathy. Biochemical and Biophysical Research Communications. 2007;362:281–287.

9.  Hu L-YR, Ackermann MA, Hecker PA, Prosser BL, King B, O’Connell KA, Grogan A, Meyer LC, Berndsen CE, Wright NT, Lederer WJ, Kontrogianni-Konstantopoulos A. Deregulated Ca2+ cycling underlies the development of arrhythmia and heart disease due to mutant obscurin. Science Advances. 2017;3:e1603081.

10. Manrai AK, Funke BH, Rehm HL, Olesen MS, Maron BA, Szolovits P, Margulies DM, Loscalzo J, Kohane IS. Genetic Misdiagnoses and the Potential for Health Disparities. New England Journal of Medicine. 2016;375:655–665.

11. Carnethon Mercedes R., Pu Jia, Howard George, Albert Michelle A., Anderson Cheryl A.M., Bertoni Alain G., Mujahid Mahasin S., Palaniappan Latha, Taylor Herman A., Willis Monte, Yancy Clyde W. Cardiovascular Health in African Americans: A Scientific Statement From the American Heart Association. Circulation. 2017;136:e393–e423.

12. Musemwa N, Gadegbeku CA. Hypertension in African Americans. Curr Cardiol Rep. 2017;19:129.

13. Kohane IS, Masys DR, Altman RB. The Incidentalome. A Threat to Genomic Medicine. JAMA. 2006;296:212–215.

14., accessed 13/05/2021.

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