The anti-S2 peptides, the stabilizer and destabilizer, were designed to target myosin sub-fragment 2 (S2) in muscle. When the peptides are coupled to a heart-targeting molecule, they can reach the cardiomyocytes and interfere with cardiac muscle contraction. Monoclonal antibodies, MF20 and MF30, are also known to interact with light meromyosin and S2 respectively. The MF30 antibody compared to anti-S2 peptides and the MF20 antibody is used as a control to test the central hypothesis that: Both the anti-S2 peptides and antibodies bind to myosin S2 with high affinity, compete with MyBPC, and possibly interact with titin, in which case the anti-S2 peptides have further impact on myosin helicity and reach the heart with the aid of tannic acid to modulate cardiomyocytes' contraction in live mice. In this research, the effects of anti-S2 peptides and antibodies on myosin S2 were studied at the molecular and tissue levels. The anti-myosin binding mechanism to whole myosin was determined based on total internal reflectance fluorescence spectroscopy (TIRFS), and a modified cuvette was utilized to accommodate this experiment. The binding graphs indicated the cooperative binding of the peptides and antibodies with high affinity to myosin. Anti-myosin peptides and antibodies competition with Myosin Binding Protein C (MyBPC) was revealed through the super-resolution expansion microscopy using wildtype skeletal and cardiac myofibrils, and MyBPC knock-out cardiac myofibril. This new emerging technique depends on using the regular confocal microscope in imaging expanded myofibril after embedding in a swellable hydrogel polymer and digestion. A decrease in the fluorescent intensity at the C-zone was observed in myofibrils labeled with fluorescently labeled anti-S2 peptides or antibodies supporting the competition with MyBPC, which further was confirmed by the absence of this reduction at the C-zone in the knockout MyBPC cardiac tissue. The anti-S2 peptide's ability to reach inside the cardiomyocytes was tested by injecting fluorescently labeled anti-S2 peptides bound to tannic acid in live mice, the destabilizer peptide reached the heart 6X more than the stabilizer peptide. Some of the peptides labeled cardiac arterioles and T-tubules as detected by super-resolution microscopic images, meanwhile some peptides reached inside the cardiomyocytes and labeled some sarcomeres. This dissertation demonstrates the ability of anti-S2 peptides and antibodies in modifying myosin as they bind cooperatively with high affinity to myosin and compete with the regulatory protein MyBPC, in addition to the possible interaction between the stabilizer peptide and titin. Lastly, the peptides succeeded in labeling some cardiac sarcomeres in live mice.
| Identifer | oai:union.ndltd.org:unt.edu/info:ark/67531/metadc2179318 |
| Date | 07 1900 |
| Creators | Quedan, Duaa Mohamad Alhaj Mahmoud |
| Contributors | Root, Douglas D., Azad, Rajeev, Benjamin, Robert C., Wang, Xiaoqiang, Padilla, Pamela A. |
| Publisher | University of North Texas |
| Source Sets | University of North Texas |
| Language | English |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Format | Text |
| Rights | Public, Quedan, Duaa Mohamad Alhaj Mahmoud, Copyright, Copyright is held by the author, unless otherwise noted. All rights Reserved. |
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