The hollow, cylindrical shape of the bodies and muscular organs of soft-bodied invertebrate animals is well suited to functions in skeletal support, movement and locomotion. This ubiquitous shape, however, may incur a previously unrecognized cost - large non-uniformities in shortening among the muscle cells arranged circumferentially around the body. Such non-uniformities pose serious problems for the muscles, connective tissues, and neural control systems of the muscular organ, and may force muscle cells in some regions of the body wall to operate with reduced mechanical efficiency during locomotion and movement. We have identified a hollow cylindrical muscular organ, the mantle of squids, in which circumferential muscle cells at different locations likely produce different amounts of force, shorten at different velocities, and generate different amounts of mechanical work during a single contraction of the tubular body. We will use morphological, electromyographic, and biomechanical approaches to investigate these potential problems and identify mechanisms in the skeletal support system of the mantle that may mitigate them. The results of our work promise to reveal new general principles of function for skeletal support systems, not just for soft-bodied invertebrates but for all animals with cylindrical muscular organs. Our research will provide opportunities for undergraduate and graduate students from diverse ethnic backgrounds to learn experimental techniques in kinematics, muscle physiology, and microscopy. Furthermore, our student participants will gain experience in the collaborative and integrative nature of science as they work in teams with peers from different institutions to collect and analyze data, and present the results of their work at national and local scientific conferences.
Our project produced exciting results with important implications for understanding muscle and connective tissue function in soft bodied invertebrates and in many hollow organs such as blood vessels. Because the wall of a hollow organ is essentially constant in volume (tissue resists volume change), when the diameter decreases, the wall thickens. This change in wall thickness means that muscle or connective tissue fibers located near the inner lumen undergo significantly greater change in length than those located near the outer surface. There is thus a previously unexamined gradient of length change present in the wall that impacts muscle and connective tissue function. We focused on the mantle of squid, which represents an ideal system to examine how the gradient of length change is accommodated. The mantle is a conical muscular tube that expands and contracts forcefully to ventilate the gills and for jet propulsion. We compared the muscle from zones near the inner and the outer surface of the mantle wall and discovered that an important contractile property, the length-tension relationship (the force of a muscle versus its length), differs at the two locations. Based on additional experiments using sonomicrometry to measure fiber length change during movement, we showed that this modulation means that all mantle muscle fibers produce approximately the same force during a cycle of mantle contraction and expansion, in spite of the gradient of length change in the mantle wall. We also discovered that the obliquely striated muscle fibers of the mantle do not exhibit the length-dependent contraction kinetics that are characteristic of vertebrate cardiac and skeletal muscle fibers, with important implications for our understanding of muscle function. We also found that the muscle fibers of the mantle all operate on the ascending limb of the length-tension curve, which means that they function at shorter lengths than that at which they produce maximum force. This is an important result because this muscle fiber type (called obliquely striated muscle) has been assumed to be specialized for operation on the descending limb of the length-tension curve. The prevailing hypothesis for the functional significance of this muscle fiber type, which is present in over a dozen animal phyla, is thus likely incorrect. We also examined a system of connective tissue fibers in the mantle of squid that function to control shape change, provide reinforcement and store elastic energy during mantle movement in order to determine how the fibers can accommodate the gradient of length change. We found that the fibers, previously designated as "Intramuscular Connective Tissue Array 3" (IM-3) accommodate the gradient of length change as a result of their arrangement; IM-3 fibers near the inner surface of the mantle are significantly more folded in a fully contracted mantle than are those near the outer surface and both become unfolded when the mantle expands. These results have important implications for connective tissue fiber reinforcement of diverse cylindrical soft-bodied animals and also for the reinforcement of blood vessels in the circulatory systems of both vertebrates and invertebrates. In addition to the studies on the mantle, the award supported an analysis, with molecular biology techniques, of the potential role of myosin isoforms in the modulation of contractile properties in squid mantle muscle and also studies of the hydrostatic skeleton of invertebrates, including the implications of change in size and burrowing locomotion. The project provided many opportunities for training undergraduate students, graduate students and postdocs. Nineteen undergraduate students, three graduate students and a postdoctoral scholar received extensive training in research. The undergraduates had the opportunity to learn diverse skills, including histology and quantitative microscopy, molecular biology techniques, muscle physiological techniques, sonomicrometry, electromyography, in addition to general lab skills and animal husbandry. The students were involved in all aspects of the research, gaining experience in searching and evaluating the literature, designing and conducting experiments, analysing results and preparing figures, and presenting data at scientific conferences. The graduate students gained experience in these areas and also in the preparation of manuscripts for publication. The postdoctoral scholar extended his training by gaining experience in the techniques of comparative biomechanics, comparative physiology and marine biology. The project provided women and underrepresented minorities with scientific training; thirteen of the participants in the study were female and two were from under-represented minority groups. We also focussed on engaging members of the general public through lectures, laboratory tours, and less formal conversations and believe that the outreach we accomplished during the grant period had a major effect. As an example, one of the undergraduates, who was a student in a fast-track teacher training program at UNC-Chapel Hill, participated in the Howard Hughes Medical Institute Summer Undergraduate Internship for Future Teachers and, based on her research in the laboratory, developed a learning module to provide curriculum support and for self-study for high school science teachers and advanced students.
