Nicholas Wallace and Dr. Laura Bridgewater, Microbiology & Molecular Biology
Bone morphogenetic proteins (BMPs) are in the transforming growth factor B (TGF-) super family of proteins, which were originally identified by their ability to induce bone formation in animals.A novel nuclear variant of BMP2 (nBMP2) was recently discovered by Dr. Bridgewater and her graduate researchers. In order to discern the exact function of this nuclear form of BMP2, nBMP2 chimeric mice were created.
Chimeric mice are formed from mice embryos. Cells are prepared with genomes lacking the nBMP2 protein and are introduced into the growing mouse embryo. As the mouse develops, the reproductive germ cells have a chance of developing from these mutant cells lacking nBMP2. In this case the offspring of the chimeric mouse should have a mutant set of genes and a normal set from the second parent. The first generation of heterozygous mice is then mated to produce litters containing pups of all three genotypes: wildtypes, heterozygotes, and mutants.
The first litter was born November 3, 2007 with each mouse’s genotype tested and recorded. Experimentson each of the ensuing litters were thenperformed to distinguish an identifiable phenotype that would differentiate the mutant mice from the wildtype. No significant differences were seen during the mice’s adolescence and early adulthood.Experiments were then performedto determine differences in balance, gait, coloring, weight, strength, and senses. No extreme variations were seen between the two groups of mice with the exception of the strength test, which showed that mutant mice experienced much quicker muscle fatigue than wildtypes. These results led to more intensive experimentation on muscle performance. Several aspects of muscle contractions were observed but only two tests gave significant results: relation rates and force frequency patterns. Both of these results indicated that the mutant mice had longer muscle relaxation time after contractions.
In order to identify the underlying cause of the differences in muscle relaxation times, I began investigating potential differences in capillary formation, muscle fiber cross-sectional area, and fiber type composition. No statistically significant variance was seen between the mutant and wildtype mice in regards to capillary formation or cross-sectional area. But after preliminary experimentation, differences were evident in the fiber type compositions. Initially, I hypothesized that an increased ratio of type II to type I fibers would be seen in the mutant mice. This would help explain the differences observed in muscle fatigue rates during limb flex strength tests. But as the research continued, the data seemed to show the opposite results.
There are several experiments that can be performed to determine the fiber type composition of skeletal muscle, but by far the most through and revealing way is to slice microscopic sections of muscle tissue and perform staining tests that will differentiate the four muscle fiber types into four varying shades of black. The results of these staining techniques can be seen in figure 1. In order to develop these images, a series of tasks must be executed with extreme caution and meticulous care. Mice are first anesthetized (and subsequently euthanized) by intraperitoneal injection of pentobarbital. The gastrocnemeus muscle are then dissected from the tircepssurare muscle group and cleared of any excess tissue. Each muscle is mounted in OCT binding compound and snap-frozen in 2-methylbutane cooled to -159° C; this not only preserves the muscle but also prevents any cell damage from ice crystal formation. The tissue blocksare subsequently placed in a 2.0 ml cryotubes and stored at -80° C. Frozen muscle samples are then serially sectioned at -20° C in a microtome cryostat. This machine producesthe extremely thin sections of muscle that are required to perform histochemical tests. Muscle fiber type distribution is then determined from adenosine triphosphatase activity at pH 9.4 after preincubation at pH 4.40. The varying pH solutions alter the proteins within the muscle fibers and allows for the staining solutions to bind at varying degrees to the four different fiber types.
Fibers were classified as type I or II based on their staining intensity (dark fibers counted as type I and light fibers counted as type II). An average of 1119 fibers wereindividually counted per mouse muscle, with a grand total of 12,304 fibers counted over the entire experiment. A videocapturing microscope and Adobe Photoshop CS3 were used to retrieve images and count specific fiber type composition of muscle sections, respectively. Analysis of retrieved data was performed with an independent t-test. Significance was set at P < 0.05. The data showed a slightly insignificant difference between the two mouse groups (P = 0.076), but the difference was still evident (figure 2). Although the data did not show significant results, the numbers still warranted further investigation. Currently I am performing SDS-page experiments to quantify the protein that is present within the muscle tissues. If present experimentation is consistent with the previous results of the histochemical staining, then mutant mice should show a significantly high amount of type I myosin heavy chain protein compared to wildtype mice. My hopes are to complete these tests by August and include them in a publication that is currently being written by Dr. Bridgewater and Dr. Hancock. The paper will contain all of the current experimental results related to nBMP2 mutant muscle performance.
Figure 2
