Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Physiology

Abstract

Free ionic calcium (Ca2+) plays an essential role as a second messenger that initiates muscle contraction in the heart. Ryanodine receptor (RyR2) acts as the primary Sarcoplasmic Reticulum (SR) Ca2+ release channel in the heart. In the event that intracellular SR Ca2+ handling is compromised, both the contractility and electrical excitability of the heart can be altered. As a consequence, heart function may not be able to maintain the necessary cardiac output to meet the metabolic demand of the body.

Increased oxidation of RyR2 has been implicated in abnormal Ca2+ handling that promotes the onset and progression of cardiovascular disease. However, little has been done to specifically characterize oxidative post-translational modifications (PTMs) of RyR2 as a result of oxidative stress within the ischemic or failing myocardium. Commonly observed in many disease phenotypes, including cardiovascular disease, is the prominence of glutathione mixed-disulfides as a result of oxidative stress. This work tests the hypothesis: Increased glutathione mixed-disulfides will promote oxidation of RyR2, promoting augmentation of SR Ca2+ fractional release and SR Ca2+ will leak. Oxidative PTMs of RyR2, observed as a result of experimentally induced oxidative stress, will manifest in models of disease.

To test these hypotheses, changes in SR Ca2+ cycling parameters were measured in isolated ventricular myocytes from rabbit. RyR2 oxidation was determined by measuring relative free thiol content and western blot analysis. In the first part, the cellular redox environment is manipulated experimentally. With increased intracellular glutathione-mixed disulfides, RyR2-mediated SR Ca2+ release and RyR2 intersubunit disulfide formation (cross-linking) was increased. Moreover, RyR2 intersubunit cross-linking was strongly associated with depleting steady state SR Ca2+. The second part defines RyR2 oxidation in ischemia-reperfused (I/R) and failing myocardium (HF). Increased RyR2 oxidation and RyR2-mediated SR Ca2+ release manifested in both I/R and HF. RyR2 intersubunit cross-linking was observable in I/R whole tissue preparations. These findings implicate intersubunit cross-linking as an important mechanism for the regulation of RyR2 channel activity in disease.

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Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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