CBET-0650686 Boriek The central hypothesis of this research is that normal diaphragm muscle tissue is anisotropic (stiffer in transverse than longitudinal direction) and this structure is: (a) mediated by desmin intermediate filaments, and (b) important to normal energy-efficient function of the diaphragm. Further, desmin may help the muscle cells to distinguish and respond differently to longitudinal and transverse strains. These questions will be answered by analyzing both normal and desmin knock-out tissue.
This project seeks to understand how cytoskeletal elements and mechanotransductive signaling mechanisms in oriented tissues give rise to anisotropy of both mechanical properties and mechanical responses in muscle tissue. These are important fundamental studies that will have broad implication on how musculoskeletal tissues are organized and tuned to sense and respond to their mechanical environment. The proposed studies involve two thrusts: experimental mechanics, and molecular cell biology. The proposed research includes several innovative aspects such as applying more physiologic, biaxial loading to diaphragm tissue and proposing the idea of directional mechanotransduction in the respiratory pump. This research will advance the understanding of respiratory pump function as well as directional mechanotransduction. The project integrates the fields of micromechanics, mechanotransduction, and respiratory physiology.
In addition, this project will enhance training of graduate students, medical students, and through a summer program (SMART) undergraduate students interested in bioengineering research. Some of these trainees will certainly be drawn from the underrepresented minority population.
(1) Intellectual Merit of the Completed Research Activities We have a long standing interest in understanding the mechanotranduction in the ventilatory pump, the diaphragm. In particular, we are interested in understanding the molecular basis for muscle anisotropy of the respiratory pump. Our in vivo and in vitro physiologic data were consistent with the diaphragm muscle being mechanically anisotropic (the property of being directionally dependent). We have published seven papers since receiving the award in 2007. These papers included mechanotransduction of respiratory muscles, mechanotransduction in smooth muscles, and in vivo physiologic study of the respiratory system. We now briefly highlight one of the main findings in one of the published manuscripts. We have investigated anisotropic regulation of Ankrd2 gene expression in diaphragm skeletal muscle. Ankrd2 is recently discovered a novel skeletal muscle specific protein. Ankrd2 belongs to the conserved muscle ankyrin repeat protein (MARP) family. Ankrd2 negatively regulates skeletal muscle growth. In this study, we identified two distinct mechanosesning signaling pathways, which are directional dependent, that regulate Ankrd2 gene expression in the diaphragm muscle. We identified that mechanical stretch in the longitudinal and transverse directions to the diaphragm muscle fibers up-regulated Ankrd2 gene expression by two distinct signaling pathways in wild-type (WT) and mdm, a mouse model of muscular dystrophy with early-onset of progressive muscle-wasting. Stretch in the longitudinal direction activated both nuclear factor kappaB (NF-kappaB) and Activator protein-1 (AP-1) transcription factors, whereas stretch in the transverse direction activated only AP-1 transcription factor. Interestingly, longitudinal stretch activated Ankrd2 promoter only by NF-kappaB, whereas transverse stretch activated Ankrd2 promoter by AP-1. Moreover, we found that longitudinal stretch activated Akt, a serine/threonine protein kinase that plays a key role in multiple cellular processes. Akt upregulated Ankrd2 expression through NF-kappaB. However, transverse stretch activated Ras-GTP, Raf-1, and Erk1/2 proteins, which up-regulated Ankrd2 expression through AP-1. Surprisingly, the stretch-activated NF-kappaB and AP-1 signaling pathways were not involved in Ankrd2 regulation at the basal level, which was high in the mdm mouse diaphragm. Taken together, our data showed the anisotropic regulation of Ankrd2 gene expression in the diaphragm muscles of WT and mdm mice via two distinct mechanosensitive signaling pathways. This study has been published in a reputed international journal, the Federation of American Societies for Experimental Biology Journal. Together, our studies provided a better understanding to an emerging novel mechanobiological paradigm; namely directional mechanotransduction. (2) Broader Impacts Resulting from the Completed Research and Educational Activities The broader impacts and societal benefits of the proposed work are significant. The proposed research has the potential to contribute to the understanding of respiratory pump function in health and disease. Muscle weakness is an enormous problem in patients with chronic pulmonary disease. Respiratory muscle weakness is also a serious problem in patients who suffer from AIDS, cancer, diabetes, trauma, burn injury or chronic heart failure. Muscle weakness has very serious negative effects upon rehabilitation and survival in those patients. Aberrant activation of some of the proposed mechanosensitive signaling pathways could lead to alteration in respiratory pump function leading to respiratory failure. Understanding the specific mechanosensitive signaling pathways that are implicated in respiratory muscle weakness could eventually help in the design of therapies to reduce loss of force production by respiratory muscles. Our findings provided critical information on the molecular mechanisms by which physiologic forces are transmitted and transduced in the respiratory pump. Our approach was to support an integrated research/educational program that tightly links the novel findings of our proposed research to the educational goals of our research program. Graduate Student Training:The completion of one of the studies on the role of titin in modulating respiratory pump function that was supported partly by this grant contributed to the advancement of the training of Mr. Michael Lopez, a graduate student at Baylor College of Medicine. Michael Lopez has completed his PhD degree in April of 2010 under the direction of Dr. Boriek. The title of his PhD dissertation was "The Respiratory pump in muscular dystrophy: Dysregulation of mechanosensitive signaling pathways." Michael has now returned to the University of Michigan Medical School to complete his MD degree requirements. It is noteworthy to state that Michael was an underrepresenteted minority student. Michael has published 6 papers with Dr. Boriek, four of which is directly related to his PhD work. Post-doctoral Research Training: Drs. Patricia Pardo, and Junaith Mohamed, postdoctoral research associates have gained particular training in respiratory muscle physiology and mechanotransduction. Their training complemented the strong background that they had in signal transduction. Taken together, our proposed integrated educational and research approach had a significant broader impact by the discovery and validation of our emerging novel mechanobiological paradigm.
