From reiser.17@osu.edu Mon Jan 14 17:13:49 2002 Date: Wed, 09 Jan 2002 08:25:51 From: Peter J. Reiser To: Savoir Faire Subject: Re: MyHC project... Hello Fred, The best thing to do is to ship a small frozen piece of each of your samples. I will trim each sample down to about 20 mg and homogenize in my sample buffer. I do not need much at all. In fact, we can analyze MHC isoform content of single cardiac myocytes. The issue becomes, with very small samples, whether it represents the whole tissue well. Therefore, it would be great if you could send a sample that is about the size of a pencil eraser. Don't worry about the shape. I suggest the eraser because it will fit into a microcentrifuge tube easily and will provide ample material. It would be nice if you could provide a sample from a consistent region (e.g., epicardium). I prefer samples shipped in labeled microcentrifuge tubes. Sharpie brand markers are the best. Alternatively, place tape on each tube and write the label with a pen. I have received samples from others that have illegible labels so that is why I am suggesting a specific method. The least preferable is samples wrapped in aluminum foil because the label inevitably becomes illegible. It would be nice if you could provide a small piece of normal pig atrium (left or right) also. It will provide a standard for MHC-alpha. I have some buried in my freezer somewhere so do not worry if you do not have it. If there is a convenient opportunity to collect an atrial sample before you ship the others, fine. Otherwise, I will dig one of my old ones out. I can also check our animal facility across the street to see if there are any terminal pigs scheduled soon. You can ship them on dry ice or with frozen ice packets. Liquid nitrogen is NOT necessary. It would be a good idea to ship them on a Monday, Tuesday or Wednesday (today would be fine) so they arrive before the weekend. Usually I receive items from others within 24 hours of shipping out. We just do not want them to sit somewhere over a weekend. Please use the following address: Peter J. Reiser Department of Oral Biology College of Dentistry 305 West 12th Avenue 4205 Postle Hall Columbus OH 43210 Phone (614) 292-8476 That should be everything you need for an overnight delivery. Please send an e-mail note when you ship them. I will then watch for them and let others know that they need to be frozen as soon as they arrive. I expect I can get everything finished within two weeks of arrival. Depending on what else I need to do at the time, it could be much shorter. Peter At 02:13 PM 01/08/2002 -0500, you wrote: > >I have 18 samples in three groups: 6 each from the control group, LVH >without clinical failure, and heart failure. Do you know the best way to >ship the samples? Frozen, after homogenization, or after calibration??? > >On Mon, 7 Jan 2002, Peter J. Reiser wrote: > >> Fred, >> >> I will certainly be happy to help. Please reply with the number of samples >> that you would like me to analyze. I can then plan how much time it will >> take to prepare, run and scan the samples. >> >> Peter >> >> >> >> At 09:59 PM 01/06/2002 -0500, you wrote: >> > >> >Hi, Dr. Reiser - >> > >> >I apologize for not getting back sooner. I've been out of town on family >> >business. I'm getting the subject data regarding weights and hemodynamic >> >numbers. It very well may be that you're hypothesis is correct, but >> >unfortunately, I need to complete this step before moving on to another >> >more interesting project. We have specimens from human transplant >> >subjects after they have been on LVAD devices for some time, and I hope to >> >examine these tissues to see if there is any normalization of MyHC >> >isoform composition in the face of improved cardiac function. Do you >> >think you would be able to help run the gels on these pig speciments? I >> >would continue endeavoring, for my part, to achieve adequate separation of >> >the alpha and beta isoforms, but I'm hoping to get some data to work with >> >by February at the latest... >> > >> >Fred >> > >> >On Wed, 26 Dec 2001, Peter J. Reiser wrote: >> > >> >> Dear Fred, >> >> >> >> I do not know with certainty but I very much expect that adult pig >> >> ventricles express essentially MHC-beta to the virtual exclusion of >> >> MHC-alpha. Here is my reasoning: I have examined hearts in at least 9 >> >> mammalian species (see Reiser and Kline, 1998, plus cat subsequent to that >> >> study). All of the small-size mammals that I have studied have significant >> >> amounts of MHC-alpha with some MHC-beta. All large mammalian species >> >> express predominantly MHC-beta. The cut-off appears to be animals that are >> >> the size of adult rabbits or cats. Cat ventricles express very little >> >> MHC-alpha. Pigs, being much larger, are likely to express even less >> >> MHC-alpha. My paper earlier this year (Reiser, Portman,...) shows that >> >> human ventricles normally express about 3-5% MHC-alpha. Miniature pigs >> >> might express a higher level of MHC-alpha in their ventricles. Therefore, >> >> if you are working with small (i.e., young) domestic pigs (around 60-80 >> >> pounds or even less) or are working with miniature pigs, you might have a >> >> considerable amount of MHC-alpha normally and a decrease with failure. My >> >> guess is that pigs that are 150 pounds or larger very likely express very >> >> small amounts of MHC-alpha in the ventricles. As was the case in our human >> >> study, I expect there would be significant alterations in the MHC-alpha >> >> levels in the atria which might not relate to your ventricular functional >> >> data but that still could be interesting. >> >> >> >> The above information on normal (non-failing) animals is consistent with >> >> earlier work of David Ianuzzo and co-workers who showed that V3 (the >> >> homodimer of MHC-beta) increases with body size. >> >> >> >> Now, I want to mention one more thing to consider. The epicardium of the >> >> ventricle may normally express significantly higher levels of MHC-alpha >> >> than a transmural sample. We find this to be especially true in small >> >> mammals and we are preparing a paper for submission. If pigs follow the >> >> same pattern and if you have samples that are primarily epicardial or if >> >> epicardial samples can still be prepared from the samples you have, then >> >> you might be able to show a change with failure in MHC isoform expression >> >> that is associated with failure. However, it might be restricted to the >> >> epicardial layer and it might not relate to a more global functional >> change. >> >> >> >> One more thing to consider is the following. If you have functional data >> >> that shows a change with failure and you do not see a large change in MHC >> >> isoform expression, that could still be very important to report. It would >> >> suggest that only a small change in myosin expression is associated with >> >> functional alterations (R. Moss and P. de Tombe had a poster at the >> >> Biophysical Society annual meeting in which they argued this point earlier >> >> this year) or it could suggest that there are other modes of adaptation, >> >> other than myosin expression, that are yielding important changes. >> >> Remember, human hearts fail without a marked change in ventricular myosin >> >> expression so if you see the same thing in pigs, it strengthens your model. >> >> >> >> I am very busy with many projects right now but I am still happy and >> >> certainly willing to help. Think it over and talk to your colleagues about >> >> my interest. Keep in mind the likelihood that large changes in ventricular >> >> MHC isoform expression will NOT be detected observed. Since the functional >> >> data was already published, it is unlikely that you would be able to >> >> publish a negative result (i.e., if there is no major change in myosin >> >> expression) by itself. >> >> >> >> Also, feel free to call me if you would like to discuss anything. I am >> >> planning on being at work most of the time except for New Year's day >> itself. >> >> >> >> Regards, >> >> Peter >> >> (614) 292-8476 >> >> >> > >> > >> > >> > > >