Single-molecule mechanics and kinetics of cardiac myosin interacting with regulated thin filaments

Abstract

The cardiac cycle is a tightly regulated process wherein the heart generates force to pump blood to the body during systole and then relaxes during diastole. Disruption of this finely tuned cycle can lead to a range of diseases including cardiomyopathies and heart failure. Cardiac contraction is driven by the molecular motor myosin, which pulls regulated thin filaments in a calcium-dependent manner. In some muscle and nonmuscle myosins, regulatory proteins on actin tune the kinetics, mechanics, and load dependence of the myosin working stroke; however, it is not well understood whether or how thin-filament regulatory proteins tune the mechanics of the cardiac myosin motor. To address this critical gap in knowledge, we used single-molecule techniques to measure the kinetics and mechanics of the substeps of the cardiac myosin working stroke in the presence and absence of thin filament regulatory proteins. We found that regulatory proteins gate the calcium-dependent interactions between myosin and the thin filament. At physiologically relevant ATP concentrations, cardiac myosin's mechanics and unloaded kinetics are not affected by thin-filament regulatory proteins. We also measured the load-dependent kinetics of cardiac myosin at physiologically relevant ATP concentrations using an isometric optical clamp, and we found that thin-filament regulatory proteins do not affect either the identity or magnitude of myosin's primary load-dependent transition. Interestingly, at low ATP concentrations at both saturating and physiologically relevant subsaturating calcium concentrations, thin-filament regulatory proteins have a small effect on actomyosin dissociation kinetics, suggesting a mechanism beyond simple steric blocking. These results have important implications for the modeling of cardiac physiology and diseases.

Scheme 1

Scheme used to interpret the rate of ATP-induced actomyosin dissociation.

Scheme 2

Scheme used to interpret attachment durations at low ATP concentrations.

Figure 1

Reconstitution of thin filaments using 10% biotinylated actin does not affect thin-filament regulation. (a) In vitro motility assay bar plots showing the speed of regulated thin-filament translocation with and without biotin-labeled actin at pCa 4, 6.25, and 9. Individual bars show the average velocity and standard deviations of >50 thin filaments measured across two separate experiments. There is no difference in speed with or without 10% biotinylated actin within each pCa value (p = 0.53, p = 0.90, p = 0.93). No movement was seen at pCa 9 with or without biotinylated actin. Representative videos can be found in Video S1. (b) Representative optical trapping traces of regulated thin filaments containing 10% biotinylated actin conducted in the presence of 1 μM ATP at low (pCa 9, red), physiologically relevant low calcium (pCa 6.25), and high calcium (pCa 4, blue). At pCa 9, binding events were very rare, and at pCa 6.25, events were qualitatively less frequent than at pCa 4 (gray lines denote binding interactions detected by the analysis program), demonstrating regulation. To see this figure in color, go online.

Figure 2

Optical trapping experiments at 1 μM ATP reveal no changes in working stroke mechanics with regulated thin filaments. Data are from 364 interactions detected from 10 myosin molecules for the unregulated condition and 491 interactions from nine myosin molecules for the regulated condition. (a and b) Representative optical trapping data traces for unregulated (black) and regulated (green) thin filaments. Gray lines indicate actomyosin interactions identified by automated event detection (see materials and methods for details). (c and d) Ensemble averages of the working stroke for myosin interacting with unregulated (left, black) and regulated (right, green) thin filaments. The rates of the exponential functions fit to the time forward (k_f) and time reverse (k_r) averages are shown along with the magnitudes of the two substeps. (e–g) Cumulative distributions derived from individual binding interactions for the (e) total displacement, (f) first substep, and (g) second substep. Each plot reports the sample mean, standard deviation, and p-values from t-tests. To see this figure in color, go online.

Figure 3

Regulatory proteins affect kinetics at low ATP due to slowing of ATP-induced dissociation. (a) Individual actomyosin attachment durations obtained in the optical trap are plotted as cumulative distributions for unregulated (black) and regulated (green) thin filaments. Distributions are fit with single exponential functions (dashed lines) to obtain the actomyosin detachment rate. Regulatory proteins significantly slow the rate of detachment (see table in d; value is the fitted rate, error is a bootstrapped 95% confidence interval, and p-value is calculated as described in the materials and methods). (b) Representative fluorescence transients from stopped-flow experiments measuring the rate of ADP release from actomyosin. Fits show single exponential fits to the average of five transients. There is no difference in the rate of ADP release measured using regulated or unregulated thin filaments (see table in d). (c) Fast phase of ATP-induced dissociation of myosin from regulated and unregulated thin filaments (see materials and methods for details). Each point represents the average of five technical repeats collected on one day. 3 separate experimental days were used. Solid lines show fitting of Eq. 1. (d) Table of parameters obtained for all kinetic measurements. The row letters indicate from which figure panel the values are derived. For the stopped-flow transient kinetics (marked “b” or “c”), the reported values are the average and standard deviations of 3 separate experimental days and p-values were calculated using a Student's t-test. To see this figure in color, go online.

Figure 4

Regulatory proteins do not affect myosin’s load-dependent kinetics at physiological ATP. (a) Representative traces collected using the isometric optical clamp. The motor bead (“M”) is moved to keep the transducer bead (“T”) at an isometric position. During a binding interaction, the average force and the attachment duration were recorded. (b) Attachment durations as a function of force measured for unregulated (black) and regulated (green) thin filaments. Each point represents a single binding interaction. 393 binding interactions were observed from 10 myosin molecules for the unregulated condition, and 611 binding interactions were observed from 20 myosin molecules for the regulated condition. Data were fitted with the Bell equation using maximum likelihood estimation to obtain k₀ and d. Error bars are the 95% confidence intervals obtained from 1000 rounds of bootstrapping simulations. These parameters were not significantly different in the presence of regulatory proteins (p = 0.16 for k₀ and p = 0.49 for d; see materials and methods for details on statistics and fitting). To see this figure in color, go online.