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(Circulation Research. 2005;96:740.)
© 2005 American Heart Association, Inc.

Cellular Biology

Functional Effects of Rho-Kinase–Dependent Phosphorylation of Specific Sites on Cardiac Troponin

Susan Vahebi, Tomoyoshi Kobayashi, Chad M. Warren, Pieter P. de Tombe, R. John Solaro

From the University of Illinois at Chicago Department of Physiology and Biophysics and Center for Cardiovascular Research College of Medicine, Chicago, Ill.

Correspondence to R. John Solaro, Department of Physiology and Biophysics (M/C 901), College of Medicine, University of Illinois at Chicago, 835 S Wolcott Ave, Chicago, IL 60612-7342. E-mail solarorj{at}uic.edu

We tested the hypothesis that activation of Rho-A–dependent kinase (ROCK-II) alters cardiac myofilament response to Ca²⁺ by mechanisms involving phosphorylation of thin filament proteins. We determined effects of a constitutively active form of ROCK-II on ATPase activity and tension development in detergent-extracted (skinned) fiber bundles isolated from mouse left ventricular papillary muscles. ROCK-II induced a depression in maximum ATPase rate and tension, which was associated with phosphorylation of troponin T (TnT), troponin I (TnI), and myosin-binding protein C (C-protein). This effect of ROCK-II was retained in fiber bundles isolated from transgenic (TG) mice in which phosphorylation sites (S14, S15, and S19) of myosin light chain 2 were mutated to alanine. Moreover, exchange of ROCK-II–phosphorylated Tn complex with the native Tn complex in the fiber bundles resulted in inhibition of maximal Ca²⁺ activation of tension and ATPase activity. Mass spectrometric analysis demonstrated that ROCK-II phosphorylated cardiac TnI (cTnI) at S23, S24, and T144 and cardiac TnT (cTnT) at S278 and T287. An important role for these cTnT sites is indicated by results demonstrating that ROCK-II induced a depression in tension and ATPase activity in skinned fiber bundles from a TG model in which cTnI is replaced by slow skeletal TnI, which lacks S23 and S24 and in which T144 is replaced by proline. Our data provide the first evidence that ROCK-II phosphorylation of the Tn complex, most likely at cTnT, has an important role in functional effects of signaling through the Rho-A pathway.

Key Words: Rho-kinase • G-proteins • heart failure • troponin T

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