Loosening the springs of the heart

Many inbred strains of laboratory rats replicate the complex symptoms found in human diseases and thus are playing an increasingly important role in biomedical research. Michael Gotthardt's group at the MDC, collaborating with Norbert Hübner's lab and scientists at the University of Madison-Wisconsin (USA), have now used such a strain to gain insights into a form of heart disease called hereditary cardiomyopathy. The study, published in the April 1 online issue of Nature Medicine, confirms that some major symptoms of the disease can be traced to a defective form of a huge muscle protein called titin. The work shows that the cause does not lie with titin itself; instead, patients' cells can't remove segments of the molecule as they produce it. This means they can't create a short form of titin that is essential to the structure and function of the heart. They also fail to process other molecules that are required for proper heart function.

This image reveals that the protein Rbm20 is found in the same location in the cell nucleus as the splicing factor U2AF65. But it does not overlap with the locations of some other splicing factors, suggesting that Rbm20 plays a role in specific types of splicing in particular cells.

Titin and other proteins begin as DNA sequences that are transcribed into RNA molecules. Most RNAs have to be processed before becoming messenger RNAs that can be translated into proteins. A crucial step along the way is alternative splicing, in which some regions of an RNA are removed. Often this can be done in several ways, meaning that one gene can give rise to protein with different parts and functions. Evolution has fine-tuned the variants to carry out specific functions in different cell types and tissues.

In the heart, for example, titin has a spring-like function that is crucial to the behavior of sarcomeres. These assemblies of proteins resemble small pistons that relax and contract during a heartbeat. When they contract, they push blood out of the heart, and they should fully expand to let the ventricles fill again. Titin helps pull back protein sheaths around the pistons to return them to a fully-extended form. If the protein is too long, it has the same effect as a limp spring. The sheaths may overextend and thus do not properly support the filling of the heart with blood. It has to work harder to nourish the body and becomes enlarged. This change in size is one feature of cardiomyopathies, which are often accompanied by arrhytmic heartbeats and other symptoms, and the result may be sudden death.

The laboratory of Marion Greaser at Madison-Wisconsin had identified a strain of rat in which some regions of titin are not removed during splicing, making it too long. One effect on the animals was the development of an enlarged heart – a symptom of human cardiomyopathies; interestingly, the rats had other symptoms as well.

"Defects in splicing are rarely caused by mutations in spliceosome proteins themselves – those are usually fatal," Michael says. "Nor did the rats have mutations in titin itself. Instead, we discovered that they were missing a region of one of their chromosomes which contained nine genes that, at the time, had not been linked to cardiomyopathy. One of them was likely the source of the problem."

When the researchers sequenced the DNA of the missing region, they discovered that the rats lacked part of a gene called RBM20. Studies of some human patients with cardiomyopathies revealed a similar mutation.

In healthy humans and animals, RBM20 protein is present in heart muscle throughout life. The molecule contains modules that normally allow it to bind to RNAs, but these parts were missing in the rats. This suggested that the protein might help a protein machine called the spliceosome recognize regions of titin RNA that need to be removed. The hypothesis would explain why rats without a working version of RBM20 couldn't process titin in the normal way.

The scientists confirmed that the rats failed to splice titin correctly only when they lacked RBM20. Would restoring the molecule change the situation? They grew cardiac cells without RBM20 in cell cultures and then introduced the molecule using a virus. Now the cells produced the short version of titin, and it functioned properly in sarcomeres.

It was unlikely that RBM20 had evolved for the sole purpose of splicing titin; maybe it affected other genes as well. Sebastian Schafer from Norbert Hübner's lab used a method called deep sequencing to study the composition of all the RNAs produced in cells without RBM20. By comparing them to RNAs taken from healthy  heart tissue, he discovered many other molecules that were spliced in a different way. Thirty-one of these cases, including titin, stood out as being strongly affected by the loss of RBM20 in both animal model and patients.

Defects in several of these molecules had already been linked to cardiac disease. The new study provided a potential connection – they might not function properly because alternative splicing wasn't able to produce molecules with the proper structure.

"The functions of these molecules include roles in the construction and behavior of sarcomeres and the transport of ions across cell membranes, which is fundamental to coordinated muscle contraction," Michael says. "We think that RBM20 serves as a specialized tool in striated muscle cells. It appears mainly in heart tissue and helps splice a number of genes that are crucial for the function of that organ. It ensures the proper construction of the heart and the establishment of a rhythmic beat by supporting communication between its cells."

- Russ Hodge

Highlight Reference:

Guo W, Schafer S, Greaser ML, Radke MH, Liss M, Govindarajan T, Maatz H, Schulz H, Li S, Parrish AM, Dauksaite V, Vakeel P, Klaassen S, Gerull B, Thierfelder L, Regitz-Zagrosek V, Hacker TA, Saupe KW, Dec GW, Ellinor PT, MacRae CA, Spallek B, Fischer R, Perrot A, Özcelik C, Saar K, Hubner N, Gotthardt M. RBM20, a gene for hereditary cardiomyopathy, regulates titin splicing. Nat Med. 2012 Apr 1.

Full text of the paper
Marion Greaser's faculty page at the University of Wisconsin