Dr. Federica Montanaro Discusses New Cardiac Dystrophin Associated Proteins: December 2012

Transcript

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Dr. Kevin Flanagan: Welcome to this month in muscular dystrophy. I’m Kevin Flanagan from the Center for Gene Therapy in Nationwide Children’s Hospital in Columbus, Ohio.

Each month on this podcast, we invite authors of recent publications to discuss how their work improves our understanding of inherited neuromuscular diseases and what their work might mean for the treatment of these diseases.

It’s my pleasure to have us our guest returning to the program today, Dr. Federica Montanaro, who’s an assistant professor of pediatrics at the Ohio State University and an investigator here at the Center for Gene Therapy.

Federica, welcome.

Dr. Federica Montanaro: Thank you for having me back.

Dr. Kevin Flanagan: We’re happy to discuss your very recent paper. To our listeners, there’s a link to her paper on our website, www.nationwidechildrens.org/muscular-dystrophy-podcast. There’s a link there to the paper published in PloS One entitled, “Proteomic analysis reveals new cardiac-specific dystrophin-associated proteins.”

That’s a bit of a mouthful but let’s start maybe with the discussion of the term, “proteomic analysis”. What does that mean?

01:11

Dr. Federica Montanaro: It refers to the proteome, which is the entire set of proteins of a sample that you’re analyzing. The proteomes are, basically, the part inside the cell that carries out different functions, so they are the little workers.

Dr. Kevin Flanagan: The machinery of the cell, in a way.

Dr. Federica Montanaro: Yes.

Dr. Kevin Flanagan: I know a lot of our listeners probably have heard words like “genome”, probably are familiar with it, that the genes contained within a cell.

Proteome, I gather, can change from cell to cell depending what those tissues are, what those cells are.

Dr. Federica Montanaro: Yes, so the proteins are encoded by the genes in the genome and they are assembled in different ways.

Dr. Kevin Flanagan: You’re looking here at the muscle proteome-- skeletal muscle proteome and cardiac muscle proteome.

Dr. Federica Montanaro: Yes but not the entire proteome of the cell; not on the entire set of proteins that the cell makes. We are interested in the proteins that are binding two dystrophin in this case.

Dr. Kevin Flanagan: How did you evaluate those?

02:12

Dr. Federica Montanaro: We’ve been using an antibody that is specific to dystrophin and this antibody only recognizes the full-length form of dystrophin.

Dr. Kevin Flanagan: An antibody is something that recognizes and holds on to a protein, is it right, a specific protein?

Dr. Federica Montanaro: Yes, it combined with very high affinity. It recognizes a particular region of a protein and so, it can be very, very highly specific with that protein.

What we’re doing is we are isolating all of the proteins from the heart tissue, and in this case, we did both heart and skeletal muscles. And then, we put the antibody in the mix, so we allow it to go in and recognize the dystrophin protein and bind tightly to it. The antibody has two parts: one part that recognizes dystrophin and another part that is able to bind to another protein called the “protein G” and we have the protein G stuck on magnetic beads.

03:10

After the antibody has bound to the proteins, we put in our magnetic beads that are coated with protein G. The protein G is going to go in, find the antibodies, and basically, these antibodies are going to start decorating this magnetic bead where they’re going to bring the dystrophin in with them.

Dr. Kevin Flanagan: That’s very cool.

Dr. Federica Montanaro: Yes.

Dr. Kevin Flanagan: You sort of fish out the dystrophin antibody and the dystrophin molecule protein associated with it.

Dr. Federica Montanaro: Yes and now, we’re just using magnet to pull out the beads.

Dr. Kevin Flanagan: That sounds very simple. That’s immunoprecipitation, I guess, what you call that.

Dr. Federica Montanaro: Yes, that is immunoprecipitation. And then, if you do it in very mild conditions, you can pull out not just dystrophin but if you’re very gentle, you can pull out also all the proteins that are binding to dystrophin in that mix.

Dr. Kevin Flanagan: I see, and that’s the co-immunoprecipitation when it comes down with it.

Dr. Federica Montanaro: Yes.

04:01

Dr. Kevin Flanagan: I see.

So really, that’s the heart of what you’ve done in this study. You look at the difference of what’s bound to the skeletal muscle dystrophin versus what’s bound to cardiac muscle dystrophin, is that right?

Dr. Federica Montanaro: It is one component of it. This is the first step, and a lot of people have been doing immunoprecipitations and the problem is you now have the proteins that come down with dystrophin. If you know what you’re looking for, then you can look at that through a technique called “Western blot”, where you then separate all the proteins that have come down with dystrophin and you look if your favorite protein is in there. But you have to know what you’re looking for and so, you can’t really make new discoveries in that sense, unless you are looking for something.

What we did was to take that mix of proteins that came down with dystrophin and apply another technique to it called “mass spectrometry” and we use a technique called “shotgun-mass spectrometry”. It’s a mouthful, but basically, this approach, you take the protein mix and proteins have a unique sequence. They sort of look like a necklace of pearls, every pearl would be an amino acid, and it’s the sequence of amino acids that dictates the sequence of the protein and the function.

05:20

There are specific proteins called “enzymes”, in this case, we used trypsin that can cut these strings of beads in particular places. They make little fragments, like little shorter strings of beads. The machine can analyze these little bits of proteins and recognize each of the little amino acids or beads and assigns them to a particular amino acid, so we can tell what is inside each of these shorter strings of beads.

Dr. Kevin Flanagan: So, you identify each little fragment, each peptide fragment.

Dr. Federica Montanaro: Yes.

Dr. Kevin Flanagan: There are millions of these each time you do this on the machine, is that right?

Dr. Federica Montanaro: Yes, there are millions of those and so, you have to use very sophisticated software to predict for each protein like for dystrophin, where would trypsin cut? What kind of little peptides would it make and then, you search for those in the mix, and you do the same things.

06:14

You scan not just for dystrophin, but for all the proteins that are known, whose sequence is known. You, basically, have a database that makes a prediction of if you were to cut any of these millions of proteins with trypsin, what their sequence, this peptides look like and you would see if they are on your pool. Basically, it’s a mix and match.

Dr. Kevin Flanagan: This is really a quite powerful technique and I’m sure our listeners can understand we couldn’t really do very efficiently just 10 or 15 years ago that we needed advances in methodology and the computer power to do this work.

Dr. Federica Montanaro: Yes.

Dr. Kevin Flanagan: Well, let’s just go back for just a minute because I know we’ll come to, in a moment, a discussion of the things that you found using mass spectrometry. But many of our listeners maybe have talked to their muscular dystrophy doctors or seen descriptions of muscular dystrophy about some things that we know that dystrophin binds to.

07:03

For example, the common diagram that we call or draw in clinic, articles show dystrophin binding to beta dystroglycan and the muscle membrane is one of its binding partners. Or to other parts of this complex of proteins called the dystrophin-associated complex, the sarcoglycans, it might be missing in limb-girdle muscular dystrophies.

So, we know about that complex. Did you find those when you did these things when you did in addition to that.

Dr. Federica Montanaro: Yes, we find them all. We find both proteins that are known to interact directly to dystrophin like the dystrobrevin, like beta-dystroglycan. We find those proteins that are known to interact indirectly with dystrophin, so they are one or two proteins removed from dystrophin.

Dr. Kevin Flanagan: Removed, you mean they don’t bind directly but bind to something that binds to dystrophin. Yes?

Dr. Federica Montanaro: Exactly. So, for example, we see alpha-dystroglycan. So, the binding is dystrophin to beta-dystroglycan, then to alpha-dystroglycan. We can see even sarcospan. Sarcospan goes through dystrophin, dystroglycan, sarcoglycans and then, sarcospan. Sarcospan might bind to other things that are middlemen.

08:11

Dr. Kevin Flanagan: Right. I know our listeners this might seems sort of confusing all these names at once, but it just sort of demonstrates in many ways what we often think of a simple model is not a simple model. There’s a lot of things that dystrophin does, that your work is going to help us sort out.

Dr. Federica Montanaro: Yes.

Dr. Kevin Flanagan: You found some surprising results in comparing skeletal muscle, the cardiac muscle. Is that fair to say, surprising results?

Dr. Federica Montanaro: Yes.

Dr. Kevin Flanagan: Can you tell us some of those?

Dr. Federica Montanaro: We found two surprising results with known members of the dystrophin-associated protein complex. The first one is in the composition of the complex for the syntrophins.

Syntrophins combine directly to dystrophin and three of them in cardiac and skeletal muscle: alpha 1, beta 1, and beta 2. The syntrophins are sort of linkers in a way. They link dystrophin to other proteins and one of them is nNOS, which is an enzyme that produces nitric oxide, which in skeletal muscle has been shown to be important for dilating the blood vessels during exercises.

09:25

Dr. Kevin Flanagan: Nitric oxide plays this role of allowing, perhaps, more blood to get into the muscles when they’re under metabolic stress.

Dr. Federica Montanaro: Yes. In the heart, it’s also involved in regulating contraction and relaxation of the heart.

Dr. Kevin Flanagan: OK, nNOS binds through syntrophins.

Dr. Federica Montanaro: nNOS binds to syntrophins and in skeletal muscle, it has been found that it depends on dystrophin for its expression at the membrane and it needs to be closer to membrane to release nitric oxide close to the membrane. So in patients with mutations in dystrophin, very often, this localization of nNOS at the membrane is lost.

10:06

What we found in the heart is that the full-length form of dystrophin that is expressed in the heart and at the membrane of the heart cells, is not binding to nNOS. We’ve done immunofluorescence to look at nNOS where it is in cardiac cells and it’s not really at the membrane. We couldn’t see that the membrane doesn’t overlap with dystrophin at the membrane.

That matches with other reports that say that nNOS is actually at places where the contractions begins, which are specific structures called T2 builds and sarcomeres, the sarcoplasm in particular.

Dr. Kevin Flanagan: The localization, so nNOS doesn’t necessarily play the same role in heart and skeletal muscle, or perhaps, doesn’t play the same exact role.

Dr. Federica Montanaro: Yes, perhaps, it’s not used in the same way and it may not need dystrophin for its localization. It doesn’t seem to need dystrophin for its localization at the sarcoplasmic reticulum in the heart.

11:02

Dr. Kevin Flanagan: This is actually of interest, I think, to some of our listeners who might know there are some trials for example, that are both on the way or other ones planned to get on the way, for drugs that modulate the nNOS pathway in patients with muscular dystrophy. So, I could see why knowing a differential localization, a differential function of nNOS, maybe important in interpreting results from a study line then.

Dr. Federica Montanaro: Yes, it could be important for the clinical trials, it could also be important to just understanding how the heart functions and how it uses nitric oxide.

Dr. Kevin Flanagan: Of course.

What else did you find that was novel or exciting to you?

Dr. Federica Montanaro: Beside nNOS as I said, we saw found the composition of the syntrophins. It was different. In particular, the heart has a beta 2-syntrophin associated with dystrophin, and in skeletal muscle, beta 2-syntrophin does not seem to bind to dystrophin. It binds to its own utrophin. So we don’t know yet the significance of this, but it might mean that it’s linking dystrophin in the heart to different cellular structure of the proteins compared to skeletal muscle.

12:15

Dr. Kevin Flanagan: Those are the comparisons of the known complex.

Dr. Federica Montanaro: Yes.

Dr. Kevin Flanagan: What did you find that was novel.

Dr. Federica Montanaro: We found that in the heart, dystrophin interacts with four other proteins and we were able to validate this interaction to show that they are happening in the mouse heart and for two of them, we were able to show that they also happen in the human heart.

The two of the proteins, Cavin-1 and Ahnak1, have some function in regulating the activity of ion channels. Ion channels are proteins that form holes in the membrane and they will allow calcium or sodium, potassium and other ions to move in and out of the cell through the port that they form.

Dr. Kevin Flanagan: They help regulate electrical activity at the cell membrane to some extent, yes.

13:03

Dr. Federica Montanaro: Yes, exactly. Problems with these channels can give arrhythmias and problems with the contractility of the heart, the rhythm of the heart, those you usually see in the clinic by abnormalities on electrocardiogram. Patients with Duchenne muscular dystrophy do show abnormalities on an electrocardiogram.

We’re hoping that the discovery that these two proteins are associated with the dystrophin in the heart might allow us what ion channels are now linked to dystrophin, and begin to understand where these electrocardiogram abnormalities are starting.

Dr. Kevin Flanagan: I see, and the other proteins you found are not ion channels ones? What do they do?

Dr. Federica Montanaro: The two other proteins are Cypher and Crystallin. Those proteins are structural proteins. They’re more involved with the contractile fibers inside the heart cell, for their integrity in the structure and linkage to the membrane. These may perform more structural roles and may be linked more to structural functions of dystrophin that are specific the heart compared to skeletal muscles.

14:19

We are just starting to investigate those possibilities. Mutations in Crystallin give rise also to dilated cardiomyopathy, and mutations in Cypher also give rise to dilated cardiomyopathy, so this may be having more an effect towards the remodeling of the heart and the loss, perhaps, of force during contraction rather than the rhythm of contraction.

Dr. Kevin Flanagan: I see. Well, this is certainly exciting work and it opens new pathways of exploration. What’s next for your group and for your lab in carrying this forward?

Dr. Federica Montanaro: For the heart part, we are eager to collaborate with cardiologists and electro physiologists for the heart to really more investigate how, as I said, in trying to identify the ion channels that interact with Cavin-1 and Ahnak1. To understand where the disease starts in the heart and are there perhaps, already any drugs available that would change the activity of those ion channels, if not, once we know what they are, that could be used and directly brought to the clinic more rapidly. That’s what we’re hoping and that’s what we’re trying to look forward for the heart.

15:35

On the other hand, we have also accumulated a lot of data on the skeletal muscle, because we have been doing this comparison, so we also now have data for skeletal muscle. We have made some very nice discoveries on that area as well that we are pursuing, that we’re hoping that this technique of immunoprecipitation coupled with mass spectrometry, we’ll be able to fill in a gap in our knowledge of how this loss of dystrophin actually lead to death of a muscle fiber or electrical abnormalities in a cardiac cell.

16:13

How does this happen? We know where it starts and we know where it ends, but we don’t know what’s happening in between. It’s sort of a black box, and so, I am hoping that we will be able to add a few pieces to that black zone right now by using this approach.

Dr. Kevin Flanagan: Well, thank you very much for sharing your work with us today.

Dr. Federica Montanaro: Thank you.

Dr. Kevin Flanagan: This podcast has been brought to you by Nationwide Children’s Hospital and by the Nationwide Children’s Wellstone Muscular Dystrophy Clinical Research Center.

You can find out more about the muscular dystrophy program and ongoing clinical trials at Nationwide Children’s at our website, www.nationwidechildrens.org/muscular-dystrophy-podcast. You’ll also find a link to the abstract of the study that we’ve discussed today.

17:02

Today, we also want to bid farewell to our executive producer for the first 20 episodes, Melissa Hamilton, who is leaving for other work. We wish you well in her new position.

Meanwhile, thanks for joining us. We look forward to next month’s podcast.

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