TY - JOUR
T1 - Small Molecule RPI-194 Stabilizes Activated Troponin to Increase the Calcium Sensitivity of Striated Muscle Contraction
AU - Mahmud, Zabed
AU - Tikunova, Svetlana
AU - Belevych, Natalya
AU - Wagg, Cory S.
AU - Zhabyeyev, Pavel
AU - Liu, Philip B.
AU - Rasicci, David V.
AU - Yengo, Christopher M.
AU - Oudit, Gavin Y.
AU - Lopaschuk, Gary D.
AU - Reiser, Peter J.
AU - Davis, Jonathan P.
AU - Hwang, Peter M.
N1 - Funding Information:
This work was funded by the Hwang Professional Corporation, which allows PMH to use his salary as a professor to pay for laboratory personnel and operating costs. PMH was previously supported by a Heart and Stroke Foundation of Canada / Mazankowski Alberta Heart Institute early career investigator award and a Canadian Institutes of Health Research Phase 2 Clinician Scientist Salary Award. NIH grant HL127699 to CMY. NIH grants R01HL132213 and R01HL138579 to JPD, and and CIHR Foundation grant to GDL.
Publisher Copyright:
Copyright © 2022 Mahmud, Tikunova, Belevych, Wagg, Zhabyeyev, Liu, Rasicci, Yengo, Oudit, Lopaschuk, Reiser, Davis and Hwang.
PY - 2022/6/8
Y1 - 2022/6/8
N2 - 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 KD 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 (KD 311 μM) in the absence of the switch region. RPI-194 acts as a calcium sensitizer, shifting the pCa50 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 (pCa50 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.
AB - 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 KD 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 (KD 311 μM) in the absence of the switch region. RPI-194 acts as a calcium sensitizer, shifting the pCa50 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 (pCa50 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.
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U2 - 10.3389/fphys.2022.892979
DO - 10.3389/fphys.2022.892979
M3 - Article
C2 - 35755445
AN - SCOPUS:85133404223
SN - 1664-042X
VL - 13
JO - Frontiers in Physiology
JF - Frontiers in Physiology
M1 - 892979
ER -