A study of tropomyosin's role in cardiac function and disease using thin-filament reconstituted myocardium

Abstract

Tropomyosin (Tm) is the key regulatory component of the thin-filament and plays a central role in the cardiac muscle's cooperative activation mechanism. Many mutations of cardiac Tm are related to hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and left ventricular noncompaction (LVNC). Using the thin-filament extraction/reconstitution technique, we are able to incorporate various Tm mutants and protein isoforms into a muscle fiber environment to study their roles in Ca(2+) regulation, cross-bridge kinetics, and force generation. The thin-filament reconstitution technique poses several advantages compared to other in vitro and in vivo methods: (1) Tm mutants and isoforms are placed into the real muscle fiber environment to exhibit their effect on a level much higher than simple protein complexes; (2) only the primary and immediate effects of Tm mutants are studied in the thin-filament reconstituted myocardium; (3) lethal mutants of Tm can be studied without causing a problem; and (4) inexpensive. In transgenic models, various secondary effects (myocyte disarray, ECM fibrosis, altered protein phosphorylation levels, etc.) also affect the performance of the myocardium, making it very difficult to isolate the primary effect of the mutation. Our studies on Tm have demonstrated that: (1) Tm positively enhances the hydrophobic interaction between actin and myosin in the "closed state", which in turn enhances the isometric tension; (2) Tm's seven periodical repeats carry distinct functions, with the 3rd period being essential for the tension enhancement; (3) Tm mutants lead to HCM by impairing the relaxation on one hand, and lead to DCM by over inhibition of the AM interaction on the other hand. Ca(2+) sensitivity is affected by inorganic phosphate, ionic strength, and phosphorylation of constituent proteins; hence it may not be the primary cause of the pathogenesis. Here, we review our current knowledge regarding Tm's effect on the actomyosin interaction and the early molecular pathogenesis of Tm mutation related to HCM, DCM, and LVNC.

Fig. 1

pCa-tension plot of the native cardiac muscle fibers and fibers reconstituted with BVC actin + Tm + Tn. (circle) Native fibers. (square) Fibers in which thin-filament was removed and reconstituted with bovine cardiac actin, Tm, and Tn. The symbols represent the data points and the curves represent best fit values to the Hill equation. (This figure is re-plotted from Bai et al. 2013)

Fig. 2

The effect of BDM on the phosphorylation status of sarcomeric proteins. (a) Pro-Q diamond stain showing phosphorylated proteins in skinned fibers, (b) Sypro Ruby stain showing the total proteins. In both a and b, left 3 lanes represent fibers skinned using the solution containing 30 mM BDM, and right 3 lanes represent fibers skinned using solution which did not contain BDM

Fig. 3

Phosphorylation analysis of sarcomeric proteins of skinned fibers from 7 different cow hearts, which were stored in the solution containing 30 mM of BDM for the specified durations (1–8 months). a Pro-Q diamond stain showing phosphorylated proteins, and b Sypro Ruby stain showing the total proteins

Fig. 4

Elementary steps of the cross-bridge cycle. Uppercase letters K indicate the association or equilibrium constants, and lowercase letters k indicate the rate constants. Collectively they are referred to as the “kinetic constants”. A actin, M myosin, D MgADP, S MgATP, and P Pi phosphate

Fig. 5

Seven periodic repeats of α-tropomyosin and the location of mutations that are known to lead to cardiomyopathies in humans. The residue number of each period's C-terminus is also indicated

Fig. 6

Nyquist plots of Tm mutants (dashed line) and WT (solid line) at 25 °C and pCa 8.0. Frequency range used is 0.13–100 Hz. The frequency increases in the clockwise direction, and the rightmost point corresponds to 100 Hz. Note the difference in scales for both axes between panel a and b. (This figure is re-plotted from Bai et al. 2011, 2012)