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Molecular mechanisms of cardiac contractility: role of myofilament proteins
xiv, 196 leaves : illustrations ; 28 cm.
ABSTRACT: Under normal physiological conditions, increasing heart rates cause the heart to contract with greater force. In addition to the calcium cycling proteins, the myofilament proteins can change its functional characteristics to support this positive force frequency relationship (FFR). Since calcium calmodulin kinase II (CaMK2) is a frequency decoder to calcium oscillations and CaMK2 phosphorylates myosin binding protein C (MyBP-C), we hypothesize that MyBP-C phosphorylation plays an essential role in changing myofilament performance characteristics in support of positive FFR. Simultaneous measurements of force and intracellular calcium concentration [Ca²⁺]in on intact mouse papillary bundle followed by a novel analysis technique that can isolate myofilament function within the context of intact muscle bundle, force/frequency measurements at physiological temperatures, and ATPase assays are used to explore the hypothesis. Increasing stimulation frequency causes increasing normalized gain of change in force to change in [Ca²⁺]in. This shows myofilaments changing their functional characteristics in a frequency-dependent manner. Inhibition of CaMK2 depresses the change in force but does not alter the change in [Ca²⁺]in. Furthermore, the data analyses reveal that CaMK2 inhibition depressed the FFR by decreasing force generation at the portion of the calcium-force loop where force increases despite monotonic decreases in [Ca²⁺]in. This segment corresponds to the effects of strongly attached crossbridges. These calcium-force results from control and CaMK2 inhibition conditions show that CaMK2 phosphorylation of MyBP-C enhances the crossbridge feedback effects to cause greater force production in a frequency dependent but calcium independent manner. Results from ATPase assays and force/frequency dependent measurements at physiological temperatures corroborate with this finding. Furthermore, a novel method using surface plasmon resonance (SPR) was developed to directly measure attachement/detachment status of crossbridges within intact sarcomeres. The SPR method was verified by using different mechanisms to vary attached crossbridge population. These mechanisms included changing [Ca²⁺], adding phosphate, adding 2,3-butanedione monoxime (BDM), and depleting adenosine triphosphate (ATP). Thus, the novel method of analyzing the force-frequency data and the application of the novel SPR method combine to form a powerful set of tools to explore the roles of myofilament proteins in molecular mechanisms of cardiac muscle contractility.
"Submitted to the Graduate School of Biomedical Sciences of The Texas A&M University System Health Science Center in partial fulfillment for the requirements for the degree of Doctor of Philosophy May 2002."
Approved as to style and content by: Mariappan Muthuchamy, David C. Zawieja, Jeremy S. Wasser, Harris J. Granger.
Ph. D. Texas A&M University System Health Science Center 2002.
Includes bibliographical references (leaves 174-195).