Genetically Engineered Addition of a Ca2+ Buffering Capacity in Mouse Cardiac Muscle Thin Filament using a Glu-Rich Segment of Insect Troponin T Alters Cardiac Performance

Abstract

In addition to a core function in Ca2+ regulation of muscle contraction, insect troponin T (TnT) has a unique glutamic acid-rich long C-terminal extension. With sequence similarity to this diverged structure, the N-terminal segment of avian flight muscle TnT binds Ca2+ with a physiologically relevant affinity (Zhang et al., Biochemistry 43:2645-55, 2004). Deletion of the C-terminal extension of Drosophila TnT significantly decreased muscle and heart functions (Cao et al., JBC 295:3794-3807, 2020). We propose a hypothesis that this structure serves as a myofilament Ca2+ reservoir to reduce the amount of Ca2+ cycled by Ca2+ pumps during muscle contraction and relaxation. A CRISP/Cas9 knock-in (KI) mouse model was constructed to replace the N-terminal variable region of cardiac TnT with 51 Glu residues derived from the C-terminal extension of bee TnT. Cardiac function is studied using ex vivo working hearts and tested for responses to beta-adrenergic stimulation or L-Ca2+ channel agonist Bay-K8644. At baseline, cTnT-51E hearts showed lower systolic velocity than that of WT control. Upon isoproterenol or Bay-K8644 treatment, cTnT-51E and wild type hearts both had positive responses in performance. However, cTnT-51E hearts exhibited significantly more increases in ventricular peak systolic pressure and diastolic velocity but still lower stroke volume as compared to that of WT controls. This phenotype is likely due to an altered response to the increase in intracellular calcium. Further analysis of Ca2+ responses of skinned muscle sections, cardiac energetic efficiency and intracellular Ca2+ transient are underway to establish the novel Ca2+ buffering capacity of TnT and its function in contractile kinetics of striated muscle.