Spectroscopic Probes of Cardiac Calcium Regulation and Therapeutic Design

Abstract

Cardiac muscle contraction and relaxation is controlled by changes in intracellular Ca2+, indicating that Ca2+ transport is a fundamental regulator of proper muscle function in the heart. The primary cardiac Ca2+ transporter is the sarcoendoplasmic reticulum Ca-ATPase 2a (SERCA2a), a transmembrane protein pump embedded in the sarcoplasmic reticulum (SR). SERCA2a pumps cytosolic Ca2+ into the sarcoplasmic reticulum (SR) of cardiac myocytes, enabling muscle relaxation during diastole. Abnormally high cytosolic [Ca2+] is a central factor in heart failure, suggesting that augmentation of SERCA2a Ca2+ transport activity could be a promising therapeutic approach. SERCA2a is inhibited by the protein phospholamban (PLB), and a novel transmembrane peptide, dwarf open reading frame (DWORF), is proposed to enhance SR Ca2+ uptake and myocyte contractility by displacing PLB from binding to SERCA2a. However, establishing DWORF’s precise physiological role requires further investigation. The work presented in this thesis focuses on the mechanisms and structural basis of SERCA2a regulation and primary drug screening of SERCA2a. In the first study (Chapter 4), we developed cell-based FRET biosensor systems that can report on protein-protein interactions and structural changes in SERCA2a complexes with PLB and/or DWORF. To test the hypothesis that DWORF competes with PLB to occupy the SERCA2a binding site, we transiently transfected DWORF into a stable HEK cell line expressing SERCA2a labeled with a FRET donor and PLB labeled with a FRET acceptor. We observed a significant decrease in FRET efficiency, consistent with a decrease in the fraction of SERCA2a bound to PLB. Surprisingly, we also found that DWORF also activates SERCA’s enzymatic activity directly in the absence of PLB at sub saturating calcium levels. Using site-directed mutagenesis, we generated DWORF variants that do not activate SERCA, thus identifying residues P15 and W22 as necessary for functional SERCA2a-DWORF interactions. This work advances our mechanistic understanding of the regulation of SERCA2a by small transmembrane proteins and sets the stage for future therapeutic development in heart failure research. In the second study (Chapter 5), we have developed fluorescence resonance energy transfer (FRET) biosensors with red-shifted fluorescent proteins, yielding improved characteristics for time-resolved (lifetime) fluorescence measurements. In comparison to biosensors with green and red FRET pairs (GFP/RFP), fluorescent proteins that emit at longer wavelengths (orange and maroon, OFP/MFP) increased the FRET efficiency, dynamic range, and signal-to-background of high-throughput screening (HTS). This combination promises to revolutionize high-precision FRET measurements from living-cells for the discovery of urgently needed therapeutics.