Theses and Dissertations
ORCID
https://orcid.org/0000-0002-7251-7661
Advisor
Johnson, Christopher
Committee Member
Fitzkee, Nicholas
Committee Member
Gwaltney, Steven
Committee Member
Davis, Jonathan
Committee Member
Emerson, Joseph
Date of Degree
5-15-2026
Original embargo terms
Embargo 2 years
Document Type
Dissertation - Open Access
Major
Chemistry
Degree Name
Doctor of Philosophy (Ph.D.)
College
College of Arts and Sciences
Department
Department of Chemistry
Abstract
The primary channel for sarcoplasmic reticulum (SR) Ca²⁺ release is the ryanodine receptor type 2 (RyR2). Calcium (Ca²⁺) signaling is the cornerstone of cardiac excitation–contraction coupling. Tight regulation of RyR2 is essential for rhythmic contractile function, while its dysregulation can underly arrhythmia and heart failure. One of the three central modulators calmodulin (CaM), Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), and calcineurin (CaN) form a regulatory triad that maintains RyR2 stability through opposing phosphorylation dephosphorylation cycles. Mutations in CaM disrupt this equilibrium and have emerged as potent drivers of inherited arrhythmia syndromes. This dissertation investigated how CaM variants reshape RyR2 regulation by kinases and phosphatases, integrating biochemical, methodological, and functional approaches. Biophysical and enzymatic analyses revealed that pathogenic CaM variants alter Ca²⁺ binding and impair CaN activation, reducing its catalytic efficiency despite preserved binding interactions. Standard methods were developed to measure Ca²⁺ sparks, waves, and SR Ca²⁺ load in isolated ventricular myocytes, enabling precise assessment of downstream effects of molecular changes. To improve accuracy, myofilament motion was pharmacologically inhibited to minimize cell contractions. High-resolution confocal line-scan imaging was utilized to achieve superior temporal and spatial detail of Ca²⁺ release events. These methodological refinements improved reproducibility and allowed evaluation across different free Ca²⁺ conditions. Functional studies revealed distinct and Ca²⁺ dependent effects of CaMKII and CaN. With wild-type CaM, CaMKII phosphorylation increased spark frequency and diastolic leak, whereas CaN promoted stabilization by modulating spark amplitude and wave activity at elevated Ca²⁺. In contrast, the F89L CaM variant amplified kinase driven RyR2 sensitization and impaired phosphatase restraint, resulting in exaggerated sparks, enhanced spark to wave transitions, and reduced SR stability. Collectively, this work advanced understanding of how CaM, CaMKII, and CaN cooperate to regulate RyR2 under controlled conditions. Also, it showed how CaM mutations destabilized this network to promote aberrant Ca2+ release events. By linking molecular defects to altered enzymatic signaling and cellular Ca²⁺ handling, these findings provided mechanistic insight into calmodulinopathy and highlight kinase–phosphatase equilibrium as an important aspect to maintain healthy cardiomyocyte function.
Recommended Citation
Afsar, Md Nure Alam, "Calmodulin integrity and kinase–phosphatase balance in the control of cardiac ryanodine receptor function" (2026). Theses and Dissertations. 6855.
https://scholarsjunction.msstate.edu/td/6855
