Structure-Function Relationships of Troponin in the Improvement of Cardiac Function

Cardiac function is vital for life. Heart failure (HF) caused by insufficient cardiac output to meet the systemic demand is the leading cause of death globally and lacks effective treatment. The contractile performance and energetic efficiency of cardiac muscle are key factors that determine cardiac function and efficiency. Cardiac muscle contraction and relaxation is regulated at the myofilament level by Ca2+ via the troponin (Tn) complex. Troponin T (TnT) is the tropomyosin (Tm) -binding subunit of Tn with a central role in anchoring Tn to thin filament and coordinating the Ca2+-modulated conformational changes. The structure and function of TnT are conserved in striated muscles of vertebrates and invertebrates. The N-terminal hypervariable region of vertebrate TnT is rich in Glu residues especially in cardiac T (cTnT) and avian flight muscle TnT. Insect TnT also has a Glu-rich long C-terminal extension. Our previous study suggested that the Glu-rich segment in the N-terminal variable region of avian pectoral muscle TnT binds Ca2+ with physiologically relevant affinity. My dissertation research will test a novel hypothesis that the Glu-rich segment segments of insect TnT, avian flight muscle TnT and cTnT may function as a myofilament Ca2+ reservoir/buffer near the Ca2+ receptor of Tn, Troponin C (TnC), for reducing the amounts of Ca2+ cycled during muscle contraction and relaxation and reducing ATP expenditure of the sarco(endo)plasmic reticulum calcium ATPase (SERCA) pumps to increase energetic efficiency of the muscle. We first studied this potentially novel mechanism in Drosophila model by deleting the Glu-rich C-terminal extension of TnT using CRISPR/Cas9 gene editing. Mutant Drosophila show significantly altered muscle and heart function, but their hearts exhibit a significantly higher tolerance to Ca2+-overloading produced by high frequency electrical pacing. It supports the myofilament Ca2+ reservoir function of the Glu-rich long C-terminal extension of insect TnT, potentially evolved for the high frequency asynchronous contraction of flight muscles. To test the hypothesis that the Glu-rich segment of TnT may function in improving the energetic efficiency of cardiac muscle in vertebrates, we constructed a CRISPR/Cas9 knock-in (KI) mouse model to replace the N-terminal variable region of cTnT with the 51 amino acid Glu-rich C-terminal extension of bee TnT. Cardiac functions were studied using isolated cardiomyocytes and ex vivo working hearts. In addition to better understand the structure-function relationship of TnT, the direct goal of my dissertation research is to explore the Ca2+ reservoir function of TnT evolutionarily selected for flight muscles as a new approach to improve cardiac muscle efficiency for the treatment of HF.