Effect of N-Terminal Extension of Cardiac Troponin I on the Ca²⁺ Regulation of ATP Binding and ADP Dissociation of Myosin II in Native Cardiac Myofibrils

Abstract

Cardiac troponin I (cTnI) has a unique N-terminal extension that plays a role in modifying the calcium regulation of cardiac muscle contraction. Restrictive cleavage of the N-terminal extension of cTnI occurs under stress conditions as a physiological adaptation. Recent studies have shown that in comparison with controls, transgenic mouse cardiac myofibrils containing cTnI lacking the N-terminal extension (cTnI-ND) had lower sensitivity to calcium activation of ATPase, resulting in enhanced ventricular relaxation and cardiac function. To investigate which step(s) of the ATPase cycle is regulated by the N-terminal extension of cTnI, here we studied the calcium dependence of cardiac myosin II ATPase kinetics in isolated cardiac myofibrils. ATP binding and ADP dissociation rates were measured by using stopped flow spectrofluorimetry with mant-dATP and mant-dADP, respectively. We found that the second order mant-dATP binding rate of cTnI-ND mouse cardiac myofibrils was three-fold as fast as that of wild type myofibrils in low Ca2+. The ADP dissociation rate of cTnI-ND myofibrils was positively dependent on calcium concentrations while the wild type controls were not significantly affected. These data from experiments using native cardiac myofibrils under physiological conditions indicate that modification of the N-terminal extension of cTnI plays a role in the calcium regulation of the kinetics of actomyosin ATPase.

SMYD2 glutathionylation contributes to degradation of sarcomeric proteins

“…Skeletal myofibrils were isolated from mouse gastrocnemius muscle. 66 Initially the frozen muscle was thawed on ice and cut into small pieces, followed by homogenization in a cold lysis buffer (10 mM Tris-HCl, pH 7.0, 5 mM EGTA, 130 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM NaN 3 , 1 mM DTT, 0.1 mM PMSF, 10 μM E-64, 100 μM leupeptin and protease inhibitor cleavage cocktail) using an electronic homogenizer. Homogenates were then pelleted at 4 °C by centrifugation for 5 min at 2,500 x g .…”

Structural Dynamics of the N-Extension of Cardiac Troponin I Complexed with Troponin C by Site-Directed Spin Labeling Electron Paramagnetic Resonance

“…The differences would also be due to the length of the synthetic peptides that were used instead of a full-length cTnI we used. In fact, there was no measurable increase in the calcium affinity of cTnC upon peptide-cTnC complex formation 16 , although the full-length cTnI increases the affinity markedly 9–12 .…”

“…The changes in the quantum yield of the fluorescence label at the residue of the N-extension of cTnI showed that it complexed with cTnC even at 0.3 M KCl 18 . Additionally, the N-extension-dependent regulation of the calcium binding affinity occurs in the actomyosin complex 9,12 or in muscle fiber 10,11 at physiological ionic strength. Upon binding their target protein, however, IDRs were assumed to lose most of their conformational freedom and adopt a well-defined structure.…”

“…However, in contrast to skeletal TnI, cardiac TnI (cTnI) possesses a unique N-extension (1–32 amino acids) that does not have a crystal structure 5 . This extension is functional for sufficient opening of the cTnC N-domain 6–8 as well as maintaining the normal Ca 2+ sensitivity of cTnC 9–12 . Furthermore, phosphorylation sites (23/24 and 42/44) for protein kinase A (PKA) and C (PKC) are present at or adjacent to this extension and modulate the Ca 2+ sensitivity after phosphorylation 4,13 .…”

Myosin and Other Energy-Transducing ATPases: Structural Dynamics Studied by Electron Paramagnetic Resonance

“…This extension is functional for sufficient opening of the cTnC N-domain [75][76][77], as well as maintaining the normal Ca 2+ sensitivity of cTnC [61,[78][79][80]. Furthermore, two phosphorylation sites (amino acid residue numbers Ser23, Ser24 and Ser42, Ser44) for protein kinase A (PKA) and C (PKC), respectively, are present in or adjacent to this extension and modulate Ca 2+ sensitivity after phosphorylation [81,82]. The structure and location of the N-terminal extension were studied by NMR, X-ray/neutron scattering, FRET, and PELDOR/DEER, but little is known because of its intrinsically disordered protein (IDP) nature [65,[83][84][85][86].…”