AUTHOR=Mahmud Zabed , Tikunova Svetlana , Belevych Natalya , Wagg Cory S. , Zhabyeyev Pavel , Liu Philip B. , Rasicci David V. , Yengo Christopher M. , Oudit Gavin Y. , Lopaschuk Gary D. , Reiser Peter J. , Davis Jonathan P. , Hwang Peter M. 

TITLE=Small Molecule RPI-194 Stabilizes Activated Troponin to Increase the Calcium Sensitivity of Striated Muscle Contraction

JOURNAL=Frontiers in Physiology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2022.892979

DOI=10.3389/fphys.2022.892979

ISSN=1664-042X

ABSTRACT=<p>Small molecule cardiac troponin activators could potentially enhance cardiac muscle contraction in the treatment of systolic heart failure. We designed a small molecule, RPI-194, to bind cardiac/slow skeletal muscle troponin (Cardiac muscle and slow skeletal muscle share a common isoform of the troponin C subunit.) Using solution NMR and stopped flow fluorescence spectroscopy, we determined that RPI-194 binds to cardiac troponin with a dissociation constant K<sub>D</sub> of 6–24 μM, stabilizing the activated complex between troponin C and the switch region of troponin I. The interaction between RPI-194 and troponin C is weak (K<sub>D</sub> 311 μM) in the absence of the switch region. RPI-194 acts as a calcium sensitizer, shifting the pCa<sub>50</sub> of isometric contraction from 6.28 to 6.99 in mouse slow skeletal muscle fibers and from 5.68 to 5.96 in skinned cardiac trabeculae at 100 μM concentration. There is also some cross-reactivity with fast skeletal muscle fibers (pCa<sub>50</sub> increases from 6.27 to 6.52). In the slack test performed on the same skinned skeletal muscle fibers, RPI-194 slowed the velocity of unloaded shortening at saturating calcium concentrations, suggesting that it slows the rate of actin-myosin cross-bridge cycling under these conditions. However, RPI-194 had no effect on the ATPase activity of purified actin-myosin. In isolated unloaded mouse cardiomyocytes, RPI-194 markedly decreased the velocity and amplitude of contractions. In contrast, cardiac function was preserved in mouse isolated perfused working hearts. In summary, the novel troponin activator RPI-194 acts as a calcium sensitizer in all striated muscle types. Surprisingly, it also slows the velocity of unloaded contraction, but the cause and significance of this is uncertain at this time. RPI-194 represents a new class of non-specific troponin activator that could potentially be used either to enhance cardiac muscle contractility in the setting of systolic heart failure or to enhance skeletal muscle contraction in neuromuscular disorders.</p>