Interaction Between Troponin and Myosin Enhances Contractile Activity of Myosin in Cardiac Muscle

Abstract

Ca2þ signaling in striated muscle cells is critically dependent upon thin filament proteins tropomyosin (Tm) and troponin (Tn) to regulate mechanical output. Using in vitro measurements of contractility, we demonstrate that even in the absence of actin and Tm, human cardiac Tn (cTn) enhances heavy meromyosin MgATPase activity by up to 2.5-fold in solution. In addition, cTn without Tm significantly increases, or superactivates sliding speed of filamentous actin (F-actin) in skeletal motility assays by at least 12%, depending upon [cTn]. cTn alone enhances skeletal heavy meromyosin's MgATPase in a concentration-dependent manner and with submicromolar affinity. cTn-mediated increases in myosin ATPase may be the cause of superactivation of maximum Ca 2þ -activated regulated thin filament sliding speed in motility assays relative to unregulated skeletal F-actin. To specifically relate this classical superactivation to cardiac muscle, we demonstrate the same response using motility assays where only cardiac proteins were used, where regulated cardiac thin filament sliding speeds with cardiac myosin are >50% faster than unregulated cardiac F-actin. We additionally demonstrate that the COOHterminal mobile domain of cTnI is not required for this interaction or functional enhancement of myosin activity. Our results provide strong evidence that the interaction between cTn and myosin is responsible for enhancement of cross-bridge kinetics when myosin binds in the vicinity of Tn on thin filaments. These data imply a novel and functionally significant molecular interaction that may provide new insights into Ca 2þ activation in cardiac muscle cells.

Micromechanical Thermal Assays of Ca2+-Regulated Thin-Filament Function and Modulation by Hypertrophic Cardiomyopathy Mutants of Human Cardiac Troponin

“…Regulated filament speed at pCa 5 (solid symbols) was at least as fast as that for unregulated F-actin (open symbols) at each temperature over the range examined ( ∼ 20–60°C) (Figure 1(c)), consistent with previous reports over more limited temperature ranges [12, 13, 16, 20]. The speed of unregulated F-actin, however, increased continuously over the entire temperature range—even at the highest temperatures examined, where regulated thin-filament speed declined.…”

Ca2+-regulatory function of the inhibitory peptide region of cardiac troponin I is aided by the C-terminus of cardiac troponin T: Effects of familial hypertrophic cardiomyopathy mutations cTnI R145G and cTnT R278C, alone and in combination, on filament sliding

“…Recombinant human α-Tm was expressed in Escherichia coli as a homodimeric fusion protein with maltose binding protein (MBP); α-Tm was purified following removal of the MBP affinity tag via thrombin cleavage as previously described [46,50,51,52,53]. After removal of the MBP tag, each of the two polypeptides in recombinant α-Tm has two extra, N-terminal amino acids (GS-); GS- is a conservative alternative to the AS-dipeptide in bacterially expressed Tm that substitutes functionally for acetylation of native Tm’s N-terminus in eukaryotic cells [54,55].…”

“…Purified Tn from human cardiac muscle (cTn) was obtained from Research Diagnostics (Flanders, NJ), or coexpressed recombinantly (rhcTn) in E. coli as a fusion protein with glutathione S-transferase (GST); the ternary rhcTn complex was purified following removal of the GST affinity tag via cleavage with TEV protease [46,50,52,56]. Human cardiac mutations of rhcTn were introduced via site-directed mutagenesis to the bacterial coexpression plasmid; changes were verified by DNA sequencing.…”

“…Control assays with unregulated F-actin were conducted in actin buffer (AB: 25 mM KCl, 25 mM imidazole, 4 mM MgCl 2 , 1 mM EGTA, 1 mM DTT, and pH 7.4) [49] with 2 mM ATP and 0.3% MC, or at pCa 5 without cTn or Tm. Motility buffer composition for assays with regulated thin filaments was calculated as described previously [46,50,52,56,60,61]. Solutions contained 2 mM MgATP, 1 mM Mg 2+ , 10 mM EGTA, sufficient Ca(CH 3 COO) 2 to achieve the desired pCa (pCa 9–4, for Ca 2+ −dependence experiments) (pCa = −log 10 ([Ca 2+ ]), where [Ca 2+ ] is in molar), 50 mM K + , 15 mM Na + , 20 mM MOPS, pH 7.00 at 30°C, 0.5% MC, and cTn and Tm (see below).…”

Slowed Dynamics of Thin Filament Regulatory Units Reduces Ca2+-Sensitivity of Cardiac Biomechanical Function

“…5,11,17,52,54,58 Actin was extracted from acetone powder prepared from rabbit back and leg muscles. 43 Rabbit skeletal myosin was prepared as described from rabbit back muscle 33 and was subjected to mild chymotryptic digestion to obtain HMM.…”