Ca2+ sensitivity of regulated cardiac thin filament sliding does not depend on myosin isoform

Abstract

Myosin heavy chain (MHC) isoforms in vertebrate striated muscles are distinguished functionally by differences in chemomechanical kinetics. These kinetic differences may influence the cross-bridge-dependent co-operativity of thin filament Ca(2+) activation. To determine whether Ca(2+) sensitivity of unloaded thin filament sliding depends upon MHC isoform kinetics, we performed in vitro motility assays with rabbit skeletal heavy meromyosin (rsHMM) or porcine cardiac myosin (pcMyosin). Regulated thin filaments were reconstituted with recombinant human cardiac troponin (rhcTn) and alpha-tropomyosin (rhcTm) expressed in Escherichia coli. All three subunits of rhcTn were coexpressed as a functional complex using a novel construct with a glutathione S-transferase (GST) affinity tag at the N-terminus of human cardiac troponin T (hcTnT) and an intervening tobacco etch virus (TEV) protease site that allows purification of rhcTn without denaturation, and removal of the GST tag without proteolysis of rhcTn subunits. Use of this highly purified rhcTn in our motility studies resulted in a clear definition of the regulated motility profile for both fast and slow MHC isoforms. Maximum sliding speed (pCa 5) of regulated thin filaments was roughly fivefold faster with rsHMM compared with pcMyosin, although speed was increased by 1.6- to 1.9-fold for regulated over unregulated actin with both MHC isoforms. The Ca(2+) sensitivity of regulated thin filament sliding speed was unaffected by MHC isoform. Our motility results suggest that the cellular changes in isoform expression that result in regulation of myosin kinetics can occur independently of changes that influence thin filament Ca(2+) sensitivity.

Figure 1. Coexpression of recombinant human cardiac troponin (rhcTn) complex

A, cDNA sequences for all three subunits of hcTn were cloned into pET 41a+ along with promotors and terminators for coexpression in E. coli. The plasmid was engineered with a GST tag at the N-terminus of hcTnT, and modified to include a TEV protease site between GST and hcTnT. B, SDS-PAGE analysis of the coexpressed rhcTn complex demonstrates that the three subunits form a complex; densitometry analysis indicates ∼96% purity. M, marker; 1, rhcTn complex after GST affinity purification and TEV protease cleavage; 2, anion exchange chromatography (DE52) void volume, containing GST tag and TEV protease; and 3–7, anion exchange chromatography fractions eluted by salt gradient. C, Western blot analysis of the coexpressed rhcTn complex. Specific antibodies for hcTnT (left), hcTnI (centre), and hcTnC (right) detected bands of the appropriate sizes for each subunit (see Methods). M, marker (MagicMark XP Western Protein Standard; Invitrogen).

Figure 2. Analysis of actin motility

A, superposition of 90 sequential video frames from one representative recorded fluorescence microscopy field. White traces indicate the paths of RhPh-labelled actin filaments over 3 s. Actin filaments in the last frame are indicated in black. Scale bar 10 μm. B, rectangular area from A magnified to illustrate measurement of the distance travelled by the trailing ends of three actin filaments. The trailing end of each actin filament is marked with a dot in the first frame and with an arrowhead in the 90th frame. Each path contour begins at the dot and proceeds to the arrowhead. Hence, the arrowheads also represent the direction of filament sliding.

Figure 3. Transmission electron micrographs of negatively stained F-actin–rhcTm–EGTA (A) and F-actin–rhcTm–rhcTn–EGTA complexes on positively charged lipid monolayers (B and C)

C is the rectangular area in B, magnified. The scale bars represent 50 nm in A, 100 nm in B and 50 nm in C. Areas of additional density on the regulated thin filaments in B and C indicate physiological binding of the rhcTn complex to actin–rhcTm.

Figure 4. Effect of recombinant human cardiac regulatory protein concentration on motility at pCa 9 (open symbols) and pCa 5 (filled symbols)

Thin filaments were reconstituted with equimolar concentrations of rhcTn and rhcTm added to flow cells (see Methods). Assays were conducted using pcMyosin (squares) or rsHMM (circles). Speeds were normalized to unregulated motility for each myosin isoform (large diamond at 0 nm rhcTn and rhcTm). Note that at concentrations ≥ 15 nm, regulatory proteins fully inhibited filament sliding at pCa 9. Also note that over the range of 15–50 nm rhcTn and rhcTm, regulated thin filament sliding speed was substantially faster than for unregulated actin.

Figure 5. Ca²⁺ sensitivity of sliding speed for regulated thin filaments propelled by pcMyosin (β-MHC, A) or rsHMM (B)

Thin filaments were reconstituted with 15 nm rhcTn and 15 nm rhcTm. Each point represents the average thin filament sliding speed (n = ∼90 filaments for 2 flow cells) at the given pCa. Error bars represent standard deviation. The curves represent non-linear least squares regression fits to the Hill Equation (eqn (1)). There was no significant difference in the regression parameter estimates of pCa₅₀ or n_H (eqn (1)) between pcMyosin and rsHMM (see Results).