Progress 10/01/10 to 09/30/15
Outputs
OUTPUTS: Our long range goal is to understand the mechanisms that regulate the onset of skeletal muscle (skm) atrophy. Our hypothesis is that the Merg1a K+ channel increases proteolysis, initiating onset of skm atrophy. The overall objective is to find safe genetic and/or pharmacological targets for enhanced skm growth and inhibition of atrophy. With this research, it may be possible to find a target for genetic selection of high muscle yield animals. Our first objective was to determine if Merg1a expression initiates UPP activity by the PI3K/Akt1/FOXO pathway and our second objective was to determine if Merg1a expression initiates UPP activity by activating the IKK-beta/kappaB-alpha/NF-kappaB pathway. We wanted to know if investigation of either pathway would present a possible safe genetic and/or pharmacological target for enhanced skm growth and inhibition of atrophy. To date, our data clearly show that the PI3K/Akt1/FOXO pathway is NOT involved in Merg1a regulation of skeletal muscle atrophy; however, the initial data suggest that the IKK-beta/kappaB-alpha/NF-kappaB pathway is involved. It may be that some aspect of the IKK-beta/kappaB-alpha/NF-kappaB pathway will prove to be a genetic target. This exciting information is being prepared for submission to a peer reviewed journal. It has been reported to the NIH as well as discussed in graduate level physiology classes on campus and in a Purdue University campus poster session. PARTICIPANTS: Individuals: Amber Pond, Principal Investigator and Research Scientist with the Department of Basic Medical Sciences within the Purdue University School of Veterinary Medicine. Partner Organization: Purdue University. Collaborators and Contacts: Drs. Ourania Andrisani, Wen-Horng Wang and Greg Hockerman (Purdue University) were collaborators on this project this past year. Training or Professional Development: Andrea Stephenson, an undergraduate pre-vet major, also worked on this project. TARGET AUDIENCES: The target audience for this work is the scientific research community, with more specific targeting of those researchers working with striated muscle. We hope to benefit animal producers and all people who consume meat and other animal products by increasing muscle yield and improving animal health. We also wish to benefit all people who are in danger of experiencing harmful levels of skeletal muscle atrophy; this would include any bedridden patients and older people who experience atrophy as a consequence of the aging process. The work may also benefit people with degenerative cardiac diseases. I have discussed this research in lectures to a Purdue University graduate class designed to discuss skeletal muscle physiology specifically. I have presented the work to a university wide research group. Further, I have spoken with an accelerated 2/3 grade class about skeletal muscle health and this project specifically (at an elementary level). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
IMPACT: We are broadening the understanding of modulation of an important proteolytic pathway. This research will identify safe genetic and/or pharmacological targets for enhanced skm growth and inhibition of atrophy; it may be possible to find a target for genetic selection of high muscle yield animals. Further, this work will allow for development of improved therapies for atrophy of both skeletal and cardiac muscle. Our 2006 paper was the first work to link the Merg1a potassium channel to protein degradation and skm atrophy. These novel data introduced a new potential target pathway for research into skeletal muscle atrophy and atrophy therapy. Further elucidation of the mechanism by which Merg1a initiates skeletal muscle atrophy will potentially enable development of high muscle yield animals and more specific and effective therapies for skeletal muscle atrophy. Higher muscle yield animals will improve animal production by increasing the profit margin per animal. Further, improved therapies for muscle atrophy will have a large social impact because these could reduce the number of cases of debilitating skeletal muscle atrophy, thereby improving the quality of life for ill and older people. Improved therapies for bedridden and elderly patients could have a huge economic impact because this would lower the cost of related patient care.
Publications
- Merg1a Expression Leads to Expression of MuRF1, but not Atrogin-1. 2011. In preparation.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: OUTPUTS: Our long range goal is to understand the mechanisms that regulate the onset of skeletal muscle (skm) atrophy. Our hypothesis is that the Merg1a K+ channel increases proteolysis, initiating onset of skm atrophy. The overall objective is to develop and characterize a muscle cell line that allows investigators to control the synthesis of the Merg1a K+ channel. Our first objective is to develop an in vitro cellular model system of C2C12 myoblasts that allows the conditional expression of Merg1a, using the tetracycline (tet) controlled expression system. This line will allow us to measure calcium currents and intracellular calcium concentrations with and without Merg1a expression; these experiments would not be possible with an in vivo model. As of last year, we had developed C2C12 lines which expressed merg1a when tet was withdrawn, however, we have determined that the expression was not tightly controlled by tet concentration; that is, epigenetic protein was present in the presence of tet. Further, the merg1a protein expression was not greatly above endogenous expression of merg1a. Therefore, this year we have changed the conditional expression approach. We are now using a "tet on" line; that is, we expect to see merg1a expression when tet is added (rather than withdrawn). We are using a newer "puro tight" expression system meant to curtail expression "leak." We have developed a number of lines which express the tet-regulator and are differentiation competent. We are now performing some transient expression experiments to ensure that we an get epigenetic expression of merg1a above the endogenous levels. We have tagged merg1a with gfp to allow us to differentiate between the endogenous and exogenous proteins. This information has been disseminated by poster presentations on campus, lectures to undergraduate/graduate physiology classes. To further examine the role of Merg1a in striated muscle, we have collaborated on studies of the effects of functional electrical stimulation (FES) on denervated muscle. We have reported (in a peer reviewed journal) that FES, indeed, can strongly curtail atrophy of skeletal muscle after denervation and, most interestingly, induce some RESCUE of atrophied muscle. We hypothesize that the FES contributes to maintenance of an ion channel population appropriate for curtailment of atrophy; that is, we believe that Merg1a synthesis is being inhibited and, therefore, UPP activity. We will pursue this hypothesis and this cell line will contribute to this endeavor. This information has been published in a peer review journal as well as discussed in graduate level physiology classes on campus. PARTICIPANTS: Individuals: Amber Pond, Principal Investigator and Research Scientist with the Department of Basic Medical Sciences within the Purdue University School of Veterinary Medicine. Partner Organization: Purdue University. Collaborators and Contacts: Drs. Ourania Andrisani, Wen-Horng Wang, Greg Hockerman (Purdue University) and Dr. Ugo Carrera (University of Padue, Italy) are/were collaborators on this project this past year. Training or Professional Development: Andrea Stephenson, an undergraduate pre-vet major, is currently working on this project. TARGET AUDIENCES: The target audience for this work is the scientific research community, with more specific targeting of those researchers working with striated muscle. We hope to benefit all people who are in danger of experiencing a great deal of harmful skeletal muscle atrophy; this would include any bedridden patients and older people who experience atrophy as a consequence of the aging process. The work may also benefit people with degenerative cardiac diseases. I have discussed this research in lectures to a Purdue University graduate class designed to discuss skeletal muscle physiology specifically. I have presented the work to a university wide research group. Further, I have spoken with an accelerated 2/3 grade class about skeletal muscle health and this project specifically (at an elementary level). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
IMPACT: We are broadening the understanding of modulation of an important proteolytic pathway. This will allow for development of improved therapies for atrophy of both skeletal and cardiac muscle. Our 2006 paper was the first work to link the Merg1a potassium channel to protein degradation and skm atrophy. These novel data introduced a new potential target pathway for research into skeletal muscle atrophy and atrophy therapy. Further elucidation of the mechanism by which Merg1a initiates skeletal muscle atrophy will enable development of more specific and effective therapies for skeletal muscle atrophy. Improved therapies for muscle atrophy would have a large social impact because these could reduce the number of cases of debilitating skeletal muscle atrophy, thereby improving the quality of life for ill and older people. Improved therapies for bedridden and elderly patients could have a huge economic impact because this would lower the cost of related patient care. Now we are characterizing a cellular model we have developed to further study the mechanism by which Merg1a induces skeletal muscle atrophy. This cell line will be shared with other interested laboratories, having the potential to open new areas of important research for many investigators. Further, the cell line will be used for work on an NIH R03 project entitled: "Role of Merg1a Potassium Channel in the Onset of Skeletal Muscle Atrophy" I was awarded this summer (June 2009) from NIAMS (National Institute of Arthritis and Musculoskeletal and Skin Diseases). Additionally, I have also collaborated with a European group who works with functional electrical stimulation (FES) to curtail muscle atrophy effects in denervated skeletal muscle. We hypothesize that the FES contributes to maintenance of an ion channel population appropriate for curtailment of atrophy; that is, we believe that Merg1a synthesis is being inhibited and, therefore, any consequent UPP activity. The group is developing a grant application to the EU (on which I am a collaborator) to explore this hypothesis.
Publications
- Roberta Squecco, Ugo Carraro, Helmut Kern, Amber L. Pond, Nicoletta Adami, Donatella, Biral,Vincenzo Vindigni, Simona Boncompagni, Tiziana Pietrangelo, Gerardo Bosco, Giorgio Fano, Marina Marini, Provvidenza M. Abruzzo, Elena Germinario, Daniela Danieli-Betto, Feliciano Protasi, Fabio Francini, and Sandra Zampieri. 2009. Sub-population of rat muscle fibers maintains an assessable excitation-contraction coupling mechanism after long-standing denervation. J Neuropathol Experimental Neurol. 68(12):pages to be assigned.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Our long range goal is to understand the mechanisms that regulate the onset of skeletal muscle (skm) atrophy. Our hypothesis is that the Merg1a K+ channel increases proteolysis, initiating onset of skm atrophy. The overall objective is to develop and characterize a muscle cell line that allows investigators to control the synthesis of the Merg1a K+ channel. Our first objective is to develop an in vitro cellular model system of C2C12 myoblasts that allows the conditional expression of Merg1a, using the tetracycline controlled expression system. This line will allow us to measure calcium currents and intracellular calcium concentrations with and without Merg1a expression; these experiments would not be possible with an in vivo model. As of last year, we had developed C2C12 cell clones which constitutively express the tet controlled transactivator protein (tTA) and then stably transfected the differentiation competent, tTA producing clone (tTA9) with the tet regulated expression vector containing the Merg1a-flag construct. This year, we have confirmed that 4 of 16 clones are differentiation competent, expressing a phenotype similar to the parent line. We have found that two of these lines express Merg1a protein in the absence of tetracycline. Additionally, we have determined that the myotubes of these two lines decrease in diameter in response to tetracycline withdrawal, suggesting that Merg1a expression induces atrophy in these cells. We are now optimizing western blotting conditions to definitively confirm the expression of Merg1a in response to tetracycline withdrawal for publication. We will begin testing for markers of atrophy in the myotubes expressing Merg1a once this is completed. This information has been disseminated by poster presentations on campus, lectures to undergraduate/graduate physiology classes and presentations to an advanced 4/5 gifted elementary class. To further examine the role of Merg1a in striated muscle, we have completed some studies of Merg1a in heart. Despite the fact that two Merg1 alternative splice variants, 1a and 1b, are known to be expressed in heart, we previously showed that only Merg1a is expressed in the atrophying skeletal muscle of mice. Now we have shown that the ratio of these alternative splice variants, 1a:1b, varies developmentally in cardiac tissue. We detected the 1a protein at a greater abundance in adult heart tissue where the in vivo cardiac Merg1 current (IKr) is almost non-detectable; however, the 1b protein is much more abundant in neonatal heart where the Merg1/IKr current is robust. This further demonstrates that tissue differences (e.g., contrast skeletal and cardiac muscle) in the Merg1 channel composition occur and suggests that different physiological consequences of Merg1 channel composition occur. It may be that Merg1a homomultimers induce UPP activity in the heart in the absence of 1b proteins, as they do in skeletal muscle. This information has been published in a peer review journal as well as discussed in graduate level physiology classes on campus. PARTICIPANTS: Amber Pond, Principal Investigator and Research Scientist with the Department of Basic Medical Sciences within the Purdue University School of Veterinary Medicine. Purdue University; American Heart Association (provided some funds for the cardiac muscle work). Drs. Ourania Andrisani, Wen-Horng Wang, Kevin Hannon, Greg Hockerman (Purdue University) and Dr. M. Borhan Alzghoul (Jordan University of Technology) are/were collaborators on this project this past year. Grant T. Abernathey, an undergraduate research assistant who is now in dental school; Andrea Stephenson, an undergraduate pre-vet major, is currently working on this project. TARGET AUDIENCES: The target audience for this work is the scientific research community at large, with more specific targeting of those researchers working with striated muscle. We hope to benefit all people who are in danger of experiencing a great deal of harmful skeletal muscle atrophy; this would include any bedridden patients and older people who experience atrophy as a consequence of the aging process. The work may also benefit people with degenerative cardiac diseases. I have discussed this research in lectures to a Purdue University graduate class designed to discuss skeletal muscle physiology specifically. I have presented the work to two university wide research groups. Further, I have spoken with an accelerated 4/5 grade class about skeletal muscle health and this project specifically (at an elementary level). Further, I met with a collaborator, Dr. David Lin from Washington State University, and we submitted an NSF grant application this summer. PROJECT MODIFICATIONS: The difficulty isolating differentiation competent clonal cells lines has delayed the progress of the project more than expected; however, we are making good progress at this point. We added some parallet studies in heart to help us further elucidate the role of Merg1a in striated muscle. These studies will further the ultimate goal of our project.
Impacts
We are broadening the understanding of modulation of an important proteolytic pathway. This will allow for development of improved therapies for atrophy of both skeletal and cardiac muscle. Our 2006 paper was the first work to link the Merg1a potassium channel to protein degradation and skm atrophy. These novel data introduced a new potential target pathway for research into skeletal muscle atrophy and atrophy therapy. Further elucidation of the mechanism by which Merg1a initiates skeletal muscle atrophy will enable development of more specific and effective therapies for skeletal muscle atrophy. Improved therapies for muscle atrophy would have a large social impact because these could reduce the number of cases of debilitating skeletal muscle atrophy, thereby improving the quality of life for ill and older people. Improved therapies for bedridden and elderly patients could have a huge economic impact because this would lower the cost of related patient care. Now we are characterizing a cellular model we have developed to further study the mechanism by which Merg1a induces skeletal muscle atrophy. This cell line will be shared with other interested laboratories, having the potential to open new areas of important research for many investigators. We have also further expanded the understanding of Merg1 channel composition in heart, contributing mechanistic information to explain observed developmental changes in current density. We have postulated a new mechanism for increases in proteolytic activity in diseased cardiac tissue. An undergraduate student, Grant Abernathey (now in dental school), participated in this work. Currently, Andrea Stephenson, an undergraduate pre-vet student, is participating in this project. She is gaining insight into basic research and muscle atrophy. I am also currently collaborating with Dr. David Lin of the University of Washington on a study of skeletal muscle physiology in hibernating brown bears. We hope to learn how bears avoid skeletal muscle atrophy during their months of inactivity.
Publications
- 1. Wang, X., R. Xu, G.T. Abernathey, J.A. Taylor, M.B. Alzghoul, K.M. Hannon, G.H. Hockerman and A.L. Pond. 2008. Kv11.1 Channel Subunit Composition Includes Mink and Varies Developmentally in Mouse Cardiac Muscle. Developmental Dynamics 237:2430-2437. (This paper was featured in the October 2008 issue of the NAVBO Vascular Biology Publications Alert.)
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Our long range goal is to understand the molecular mechanisms that regulate the onset of skeletal muscle (skm) atrophy. Our hypothesis is that the Merg1a K+ channel increases proteolysis, initiating onset of skm atrophy. The overall objective is to develop and characterize a muscle cell line that allows investigators to control the synthesis of the Merg1a K+ channel. Our first objective is to develop an in vitro cellular model system of C2C12 myoblasts that allows the conditional expression of Merg1a, using the tetracycline controlled expression system. To date, we have successfully developed C2C12 cell clones which constitutively express the tet controlled transactivator protein, tTA. We isolated 15 clones and have screened 11 for differentation competence, discovering only three clones that are differentation competent. Only one of these clonal lines differentiates in a manner consistent phenotypically with the parent line. It is not likely that tTA expression interferes
with differentiation. This event is a product of the clone development process. That is, differentiation incompetent C2C12 cells (naturally a part of the C2C12 cell line) are more hardy and most easily transfected; therefore, any tTA positive cells are more likely to be differentiation incompetent. Further, we have engineered a flag-tagged Merg1a construct and cloned it into the tet regulated expression vector (pUHD10-3). We cloned a triple flag tag onto the C-terminus of the Merg1a sequence (rather than the myc tag) because the larger flag tag will allow us to more easily distinguish between this (larger) recombinant protein and the native Merg1a on western analysis with Merg1 antibody. We hope to use the one Merg1 antibody rather than the Merg1 antibody plus a flag antibody to detect, and distinguish between, Merg1a and Merg1a-flag. The cloned insert has been sequenced and we have begun confirmation of the functional properties of Merg1a-flag protein by standard electrophysiological
approaches. Additionally, we have stably transfected the differentiation competent, tTA producing clone (tTA9) with the tet regulated expression vector (pUHD10-3) containing the Merg1a-flag construct and are currently working with 16 clones, five of which we have already confirmed as differentiation competent. We will test each of these for for tet-regulated Merg1a expression by western blot analysis (of whole cell extracts grown +/-tet) with both Merg1 and flag-tag antibody initially. Further, we have provided stronger evidence to support the link between Merg1a expression and the ubiquitin proteasome proteolytic (UPP) pathway, responsible for ~80% of the protein degradation that occurs during skeletal muscle atrophy. We have shown that mRNA levels for two different skeletal muscle specific ligases (Murf1 and Atrogin-1), active in the UPP pathway, are upregulated in response to ectopic Merg1a expression. Also, we have used an atrogin-1 luciferase reporter plasmid to show that the
atrogin-1 promoter is activated in response to Merg1a expression. These data will be offered as preliminary data for an NIH R03 submission in February.
PARTICIPANTS: Amber Pond, PhD: Principal Investigator and Research Scientist with the Department of Basic Medical Sciences within the Purdue University School of Veterinary Medicine. Two undergraduate research assistants, both desiring careers in the health profession, are working on this project. They are gaining insight into research and basic science. Claire Walther: Undergraduate Research Assistant and pre-vet major. Alan Yaacoub: Undergraduate Research Assistant and pre-med major.
TARGET AUDIENCES: The target audinece for this work is the scientific research community at large, with more specific targeting of those researchers working with skeletal muscle. We hope to benefit all people who are in danger of experiencing a great deal of harmful skeletal muscle atrophy; this would include any bedridden patients and older people who experience atrophy as a consequence of the aging process. This information was published (as reported in last year's report). Since then, I have discussed this research in lectures to a Purdue University graduate class designed to discuss skeletal muscle physiology specifically. I have presented the work to two university wide research groups. Further, I have spoken with an accelerated fourth grade class about skeletal muscle health and this project specifically (at an elelmentary level).
Impacts
Our 2006 paper was the first work to link the Merg1a potassium channel to protein degradation and skm atrophy. These novel data introduced a new potential target pathway for research into skeletal muscle atrophy and atrophy therapy. Further elucidation of the mechanism by which Merg1a initiates skeletal muscle atrophy will enable development of more specific and effective therapies for skeletal muscle atrophy. Improved therapies for muscle atrophy would have a large social impact because these could reduce the number of cases of debilitating skeletal muscle atrophy, thereby improving the quality of life for ill and older people. Improved therapies for bedridden and elderly patients could have a huge economic impact because this would lower the cost of related patient care. Now we are developing a cellular model to further study the mechanism by which Merg1a induces skeletal muscle atrophy. Once developed, this cell line will be shared with other interested
laboratories, having the potential to open brand new areas of important research for many investigators. Two undergraduate students (a pre-vet and a pre-med student) are currently participating in this project, gaining insight into research and muscle atrophy. These potential medical professionals are gaining a better understanding of basic science and research. I am also currently communicating with Dr. David Lin of the University of Washington about potential collaboration on a study of skeleltal muscle physiology in hibernating brown bears.
Publications
- No publications reported this period
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Progress 10/01/05 to 09/30/06
Outputs
Our long range goal is to understand the molecular mechanisms that regulate the onset of skeletal muscle (skm) atrophy. Our hypothesis is that the Merg1a K+ channel increases proteolysis, initiating onset of skm atrophy. The overall objective of this application is to develop and characterize a muscle cell line that allows synthesis of the Merg1a K+ channel to be turned off and on. Initial Work: To further demonstrate that Merg1a is a potential therapeutic target for skm atrophy, we treated mice experiencing skm atrophy with the Merg1 channel blocker, astemizole. Mice were gavaged orally with either: water or 160 mg/kg astemizole (as determined by dose-response studies) in water. One group of each water and drug treated mice remained weight (wt) bearing while one group of each was hindlimb suspended. Astemizole treatment alone had no significant effect on body wt. However, suspension caused a decrease in body wt that was significantly alleviated by drug treatment.
Importantly, the decrease in atrophied skm fiber cross sectional area (csa) was blocked by drug treatment, bringing fiber csa values to control levels. Interestingly, astemizole treatment produced significant increases in fiber csa, gastrocnemius muscle wt to body wt ratio and absolute gastrocnemius muscle wt in wt bearing mice. These results are meaningful because they show that block of Merg1 alleviates disuse atrophy and results in skm hypertrophy. Also, because Merg1 protein is also expressed in heart, skm specific therapies will have to be developed. We have shown that the heart Merg1 channel is composed of two different alpha subunit alternative splice variants (1a and 1b) while the skm Merg1 channel is composed solely of 1a. Therefore, we used real time rt-PCR analysis to compare Merg1a expression levels in heart with those in control and atrophied skm. The data show that there are similar levels of Merg1a gene expression in heart and atrophied skm. These results are meaningful
because they demonstrate that tissue specific therapy will not be able to exploit differences in tissue specific expression of Merg1a. Objective 1 is to develop an in vitro cellular model system of C2C12 myoblasts that allows the conditional expression of Merg1a, using the tetracycline controlled expression system. To reach this objective, we have successfully cultured C2C12 myoblasts and differentiated these into myotubes as determined by western blot analysis of cell extracts for myosin heavy chain, a marker of late stage myotube differentiation. Also, we have used western blot analysis to detect Merg1a protein in C2C12 myoblast and myotube extracts. The obvious next step is to determine relative abundances of Merg1a protein in myoblasts and myotubes. Taken together, all additional data show that Merg1 can be blocked pharmacologically, but a skeletal muscle specific therapy will need to be developed. Also, we are able to culture C2C12 myoblasts and terminally differentiate myotubes
and detect Merg1 protein in both.
Impacts
This study is the first work to link Merg1a to protein degradation and skm atrophy. The data show that the presence of the erg1a potassium ion channel in skeletal muscle increases protein degradation and induces skeletal muscle atrophy; indeed, pharmacological and genetic block of this channel inhibit skeletal muscle atrophy. These novel data delineate a new potential target for skeletal muscle atrophy therapy. Further elucidation of the mechanism involved will enable development of more specific and effective therapies for skeletal muscle atrophy. Use of this information has the potential to have a large social impact because it could reduce the number of cases of debilitating skeletal muscle atrophy, thereby improving the quality of life for ill and older people. Improved therapies for bedridden and elderly patients could have a huge economic impact because it would lower the cost of related patient care.
Publications
- Wang X. Hockerman GH. Green HW 3rd. Babbs CF. Mohammad SI. Gerrard D. Latour MA. London B. Hannon KM. Pond AL. Merg1a K+ channel induces skeletal muscle atrophy by activating the ubiquitin proteasome pathway. FASEB Journal. 20(9):1531-3, 2006 Jul.
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