Heart disease is the leading cause of death throughout the world and one of the major hallmarks is dysfunctional muscle contractility. Contractility is a highly regulated and complex process which involves multiple proteins. This network can be easily disrupted by mutation or changes in protein expression level and thus, proteins involved in this process are key drug targets. Muscle contractility is controlled by the calcium concentration in the cytosol. In cardiomyocytes, the sarco(endo)plasmic reticulum Ca2+ -ATPase, SERCA, and its regulatory protein, phospholamban (PLN) are responsible for ~70% of Ca2+ reuptake into the SR. While unphosphorylated, PLN inhibits SERCA by lowering its apparent Ca2+ affinity. Upon phosphorylation by PKA at Ser16, PLN inhibition is relieved. Mutations or disruptions in this complex have found in many forms of heart disease; thus, understanding the molecular interactions between SERCA and PLN, along with possible regulatory molecules, is essential. Understanding the molecular mechanisms that occur on a beat-to-beat basis will be essential for developing therapeutics to treat cardiomyopathies. In this dissertation work, I studied the structural, biochemical and biophysical properties of SERCA and PLN. We found that single-stranded DNA (ssDNA), RNA, and DNA analogs bind the cytoplasmic domain of PLN with low nanomolar dissociation constants, relieving inhibition of SERCA. The relief of inhibition is length-dependent, while affinity is constant for oligonucleotides longer than 10 bases. Solution and solid-state NMR experiments have provided residue specific information that ssDNA targets the cytoplasmic domain of PLN and does not affect SERCA in the absence of PLN. In-cell FRET and NMR experiments determined that addition of ssDNA does not dissociate PLN from SERCA. Additionally, I started moving this work into a sustainable, in vivo system using cardiomyocytes derived from induced pluripotent stem cells (iPSCs) to investigate the functional effects of these molecules with PLN in cell. The establishment of this cell line in our laboratory will allow for future characterization of not only XNAs with PLN, but also experiments with any cell type obtainable through differentiation of iPSCs. Finally, early work with the R14del hereditary mutant of PLN helped to determine the structural changes this mutant imposes on PLN as well as when in complex with SERCA. We found that the R14del mutant is loss-of-function and also if phosphorylated, will not relieve inhibition of SERCA. We believe that the knowledge gained here on the SERCA-PLN complex contributes to the overall understanding of how calcium handling can be modulated to change protein function and demonstrates a novel avenue of oligonucleotide action in the body. miRNAs may have evolved to also directly interact with non-transcription related proteins to modulate their function. These results and our future experiments will provide a promising avenue for development of novel therapeutic regulators of the SERCA-PLN complex to help treat heart disease, as well as provide some of the needed mechanistic insight on how hereditary mutants like R14del cause aberrant regulation in the heart.
University of Minnesota Ph.D. dissertation. May 2017. Major: Chemistry. Advisor: Michael Bowser. 1 computer file (PDF); xix, 180 pages.
Tuning the Equilibrium: A Biophysical Approach to Controlling Cardiac Contractility through SERCA and Phospholamban.
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