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Effect of Post-translational Modification Crosstalk on Thin Filament Regulatory Function in Cardiac Muscle

Nixon, Benjamin R
http://rave.ohiolink.edu/etdc/view?acc_num=osu1397558646

Abstract Details

2014, Doctor of Philosophy, Ohio State University, Integrated Biomedical Science Graduate Program.
Heart disease, with an anticipated $316 billion in economic expenses, affects one in three adults and is the leading cause of death in the United States. In the diseased heart a multitude of cellular changes occur, either as a compensatory mechanism to deter the modifications brought on as a result of cardiac disease or a direct result of the pathophysiology. One such avenue for uncovering the molecular mechanisms underlying cardiac disease and their functional changes is to study post-translational modification (PTM) of proteins. While extensive work has been done characterizing phosphorylation of cardiac contractile regulatory proteins, this work has been conducted investigating the modifications in isolation. Despite the fact that a single phosphorylation site may be sufficient to alter function, the additive functional effect of multiple phosphorylation sites to crosstalk inter-molecularly and change function differently than that evoked by a single phosphorylation must be taken into consideration. Initially, we sought to determine if the metabolic regulatory kinase AMPK phosphorylates cardiac troponin I (cTnI) at Ser-150 in vivo to alter cardiac contractile function directly at the level of the myofilament. Rabbit cardiac myofibrils separated by two-dimensional isoelectric focusing subjected to a Western blot with a cTnI phosphorylation-specific antibody demonstrate that cTnI is endogenously phosphorylated at Ser-150 in the heart. Treatment of myofibrils with the AMPK holoenzyme increased cTnI Ser-150 phosphorylation within the constraints of the muscle lattice. Compared with controls, cardiac fiber bundles exchanged with troponin containing cTnI pseudo-phosphorylated at Ser-150 demonstrate increased sensitivity of calcium-dependent force development, blunting of both PKA-dependent calcium desensitization, and PKA-dependent increases in length dependent activation. We next wanted to investigate the effect of ischemic pH on Ser-150 and Ser-23/24 phosphorylation. We demonstrate phosphorylation of cTnI is simultaneously increased at Ser-150 and Ser-23/24 during in vivo myocardial ischemia. Myocardial ischemia is known to decrease intracellular pH directly resulting in depressed Ca2+ binding to Tn and impaired contraction. To determine the pathological relevance of these simultaneous TnI phosphorylations in ischemia we measured the individual effects of TnI Ser-150 (S150D), Ser-23/24 (S23/24D) or their combined (S23/24/150D) pseudo-phosphorylation on thin filament regulation at acidic pH similar to that in myocardial ischemia. Results demonstrate that while acidic pH decreased thin filament Ca2+ binding of all TnIs, TnI S150D attenuated this decrease such that it was similar to non-phosphorylated TnI at normal pH. The dissociation of Ca2+ from troponin C (TnC) was unaltered by pH, such that TnI S150D remained slow, S23/24D remained accelerated and the combination of Ser-150 and Ser-23/24 pseudo-phosphorylation on the same TnI molecule retained accelerated dissociation. Lastly, to investigate the remaining component of thin filament regulation, tropomyosin nitration or phosphorylation was investigated to determine the effect of PTM on structure and function. To investigate the kinetic regulatory role of aTm phosphorylation we expressed and purified native N-terminal acetylated Ser-283 wild-type, S283A phosphorylation null and S283D pseudo-phosphorylation Tm mutants in insect cells. Purified Tm's regulate thin filaments similar to that reported for muscle purified Tm. Steady-state Ca2+ binding to TnC in reconstituted thin filaments did not differ between the three Tm's, however dissociation of Ca2+ from filaments containing pseudo-phosphorylated Tm was slowed compared to wild-type Tm. Replacement of pseudo-phosphorylated Tm into myofibrils similarly prolonged the slow phase of relaxation and decreased the rate of the fast phase without altering activation kinetics. Additionally, we sought to investigate the effect of reactive nitrogen species to nitrate Tm Tyr residues, its structure-function impact and develop a mass spectrometry approach to identify Tm 3-nitotyrosin (3-NT) PTM. Our data demonstrates the pathologically relevant reactive nitrogen species peroxynitrite modifies Tm Tyr residues to 3-NT with structural impact significant to modulate Tm function. We further developed and validated a novel and highly versatile target-driven MS/MS strategy to facilitate identification and quantification of Tm 3-NT without a priori knowledge of target residue modification. In conclusion, the structural and functional modification of thin filament regulatory proteins described above provide an intricate glimpse into the mechanisms in which the heart can undergo to alter function both in normal physiology and in disease states. Gaining a better understanding of protein PTMs associated with cardiac disease will play a major role in the continued development of therapeutics to treat cardiovascular disease.
Brandon Biesiadecki, PhD (Advisor)
Jonathan Davis, PhD (Committee Member)
Michael Freitas, PhD (Committee Member)
Mark Ziolo, PhD (Committee Member)
221 p.
Biochemistry; Biomedical Research; Biophysics
cardiac; troponin; TnI; phosphorylation; muscle regulation

Recommended Citations

Citations

  • Nixon, B. R. (2014). Effect of Post-translational Modification Crosstalk on Thin Filament Regulatory Function in Cardiac Muscle [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397558646

    APA Style (7th edition)

  • Nixon, Benjamin. Effect of Post-translational Modification Crosstalk on Thin Filament Regulatory Function in Cardiac Muscle. 2014. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1397558646.

    MLA Style (8th edition)

  • Nixon, Benjamin. "Effect of Post-translational Modification Crosstalk on Thin Filament Regulatory Function in Cardiac Muscle." Doctoral dissertation, Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397558646

    Chicago Manual of Style (17th edition)

osu1397558646
138
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This open access ETD is published by The Ohio State University and OhioLINK.