Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions

Abstract

Familial hypertrophic cardiomyopathy (FHC) is the most common cause of sudden cardiac death in young individuals. Molecular mechanisms underlying this disorder are largely unknown; this study aims at revealing how disruptions in actin-myosin interactions can play a role in this disorder. Cross-bridge (XB) kinetics and the degree of order were examined in contracting myofibrils from the ex vivo left ventricles of transgenic (Tg) mice expressing FHC regulatory light chain (RLC) mutation K104E. Because the degree of order and the kinetics are best studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs in an ex vivo ventricle was minimized to ∼20. Autofluorescence and photobleaching were minimized by labeling the myosin lever arm with a relatively long-lived red-emitting dye containing a chromophore system encapsulated in a cyclic macromolecule. Mutated XBs were significantly better ordered during steady-state contraction and during rigor, but the mutation had no effect on the degree of order in relaxed myofibrils. The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. The stopped-flow experiments revealed a significantly faster observed dissociation rate in Tg-K104E vs. Tg-wild-type (WT) myosin and a smaller second-order ATP-binding rate for the K104E compared with WT myosin. Collectively, our data indicate that the mutation-induced changes in the interaction of myosin with actin during the contraction-relaxation cycle may contribute to altered contractility and the development of FHC.

interactions of myosin with actin and associated proteins (sarcomeric proteins) are responsible for muscle contraction (19, 24, 48). These interactions include the mode of attachment of actin to myosin during different physiological states and the kinetics of conformational changes of myosin when interacting with actin. Familial hypertrophic cardiomyopathy (FHC) has been recognized to be caused by mutations in all major sarcomeric proteins of the heart, including the myosin regulatory light chain (RLC) (1). Compared with the myosin heavy chain, mutations in the RLC are rare, but often associated with malignant outcomes (18, 27, 41). It is therefore important to find out whether these FHC-linked mutations result in alterations of the mode of attachment of myosin to actin, and/or whether they lead to alterations of kinetics of acto-myosin interactions. These changes may ultimately contribute to the development of FHC. In this report we have focused on the K104E (lysine to glutamic acid) substitution in the myosin RLC expressed in the heart of transgenic (Tg) mice (25). The mutation was identified in a Danish population by Andersen et al. (2) and was associated with left ventricular and septal hypertrophy and diastolic filling abnormalities.

In the three-dimensional RLC structure, the residue K104 is close to both the Ca²⁺-Mg²⁺ binding loop and to the myosin light chain kinase-specific serine-15 phosphorylation site (45). It is therefore possible that this single-point mutation results in alteration of the order with which myosin cross bridges (XBs) interact with actin filaments. Furthermore, because the lever arm and the catalytic domain of a XB are in constant communication (46), the malfunction of the RLC caused by K104E may affect the acto-myosin interaction, most critically the force production and the rates of force development and relaxation. In this report we aimed at measuring the effect of the K104E mutation on the degree of order of XBs in a small observational volume containing fluorescently labeled myofibrils prepared from previously generated Tg-K104E mice expressing the human ventricular RLC-K104E in the heart (25).

The degree of order and XB kinetics are best studied when an individual XB makes a significant contribution to the overall signal, because the mean values of a large number of independent random measurements, each with a well-defined expected value and well-defined variance, are approximately normally distributed (47). Therefore, because the whole ventricle contains at least 10¹⁸ molecules of actin or myosin (left ventricle of our B6 mice typically weighs 30 mg), there is no hope of observing differences between spatial distributions of XBs within ventricles. On the other hand, if only a few XBs are observed, the differences in distribution become clear.

An argument that a small number of molecules must be studied when measuring XB kinetics is different: force-generating molecules are unsynchronized, and macroscopic measurements generate only the average values. However, when only a few molecules are studied, the value of a variable is affected by fluctuations around the average (16, 17, 44). The relative size of fluctuations depends on the number of molecules under observation N as $\sqrt{N}/N$. Even if “only” a million XBs were studied, the fluctuation would have been 0.1%. In our experiments, a typical number of molecules was 20, and the corresponding fluctuations were ∼20%. The alternative is to apply rapid transients (11, 26) to synchronize XBs. In the present work we used both approaches.

We report here that the K104E mutation in RLC imposed alterations in the degree of order and kinetics of myosin XBs in contracting myofibrils from the left ventricle of Tg-K104E mice. The mutation resulted in a significantly better order of XBs during steady-state contraction and during rigor, but it had no effect on the degree of order in the relaxed myofibrils. These changes paralleled significantly increased rates of XB binding to the thin filaments and the rate of execution of the power stroke. The stopped-flow experiments revealed significantly faster observed dissociation rates (k_obs) in Tg-K104E vs. Tg-wild-type (WT) myosin at low MgATP concentrations, and the linear regression analysis of the k_obs-MgATP concentration ([MgATP]) dependence showed a smaller second-order ATP-binding rate for the K104E compared with WT myosin.

MATERIALS AND METHODS

Chemicals and solutions.

All chemicals were from Sigma-Aldrich (St. Louis, MO). The dye SeTau-647-monomaleimide was from SETA BioMedicals (Urbana, IL). Glycerinating solution contained 50% glycerol, 150 mM KCl, 10 mM Tris·HCl, pH 7.5, 5 mM MgCl₂, 5 mM EGTA, 5 mM ATP, 1 mM DTT, 2 mM PMSF, and 0.1% β-mercaptoethanol. Rigor solution contained 50 mM KCl, 10 mM Tris·HCl, pH 7.5, and 2 mM MgCl₂. EDTA-rigor contained 50 mM KCl, 10 mM Tris·HCl, pH 7.5, and 5 mM EDTA. Ca-rigor contained 50 mM KCl, 10 mM Tris·HCl, pH 7.5, 2 mM MgCl₂, and 0.1 mM CaCl₂. Contracting solution contained 50 mM KCl, 10 mM Tris·HCl, pH 7.5, 5 mM MgCl₂, 0.1 mM CaCl₂, 5 mM ATP, 20 mM creatine phosphate, and 10 U/ml of 1 mg/ml creatine kinase. Relaxing solution contained 50 mM KCl, 10 mM Tris·HCl, pH 7.5, 5 mM MgCl₂, 5 mM ATP, and 2 mM EGTA.

Preparation of mouse cardiac myosin and glycerinated left ventricular muscle strips.

Myosin was isolated from the left (LV) and right (RV) ventricles of Tg-K104E and Tg-WT mice as described in Kazmierczak et al. (28). Briefly, after death, whole hearts were isolated, and the atria were removed. LV and RV were flash-frozen and stored at −80°C until processed. The ventricular tissue was later thawed in an ice-cold Guba Straub-type buffer (pH 6.5) consisting of 300 mM NaCl, 100 mM NaH₂PO₄, 50 mM Na₂HPO₄, 1 mM MgCl₂, 10 mM EDTA, 0.1% NaN₃, 10 mM Na₄P₂O₇, 1 mM DTT, and 1 μl/ml protease inhibitor cocktail (Sigma-Aldrich) in a volume of 0.75 ml buffer/0.2 g tissue. Ventricles kept on ice were first minced by hand and then homogenized for 2 min at a frequency of 30 Hz in a Mixer-Mill MM301 (Retsch). The homogenate was then incubated on ice for 40 min before centrifugation for 1 h at 200,000 g. The supernatant was then diluted 60-fold (by volume) with 2 mM DTT and incubated on ice for 1 h. The samples were centrifuged again for 10 min at 8,000 g, and the pellets were then resuspended in a minimal volume of buffer containing 0.4 M KCl, 10 mM MOPS (pH 7.0), 5 mM DTT, and 1 μl/ml protease inhibitor cocktail. Samples were then diluted 1:1 with glycerol, mixed gently, and stored at −20°C for up to ∼10 days. On average, one myosin preparation was obtained from a pool of approximately five hearts. All animal studies were conducted in accordance with the University of Miami Miller School of Medicine institutional guidelines, and we have received required Institutional Animal Care and Use Committee approval. Muscle strips were prepared from flash-frozen hearts of Tg-WT mice (52) and Tg-K104E mice (25). LV were isolated and dissected into small muscle bundles in a cold room in ice-cold pCa 8 solution containing 30 mM butanedione monoxime and 15% glycerol (42). They were then chemically skinned in 50% glycerol and 50% pCa 8 solution {10⁻⁸ M Ca²⁺ concentration, 1 mM free Mg²⁺ concentration [total MgPr (propionate) = 3.88 mM], 7 mM EGTA, 2.5 mM [MgATP], 20 mM MOPS, pH 7.0, 15 mM creatine phosphate, and 15 U/ml of phosphocreatine kinase, ionic strength = 150 mM adjusted with KPr} containing 1% Triton X-100 for 24 h at 4°C. Mouse LV muscle bundles were then transferred to the same solution without Triton X-100 and shipped to the laboratory in Fort Worth, TX (Borejdo).

Preparation and cross-linking of myofibrils.

To observe a few XBs, it is preferable to observe myofibril preparations rather than isolated myocytes. Myofibrils are much thinner (∼0.5 μm) than the depth-of-view of a confocal microscope (∼3 μm) with the result that the volume from which fluorescence is detected (hence the number of XBs) is smaller than if the fluorescence was collected from the entire confocal volume (This procedure reduces the number of observed XBs from thousands to 20–40.). In addition, because they are much thinner than myocytes, they are easier to penetrate by fluorescent peptide and thus prevent the problems associated with the nonuniformity of labeling of sarcomeres. To prepare myofibrils, LV was washed three times with ice-cold EDTA-rigor solution for 30 min to wash out ATP present in the glycerinating solution without causing contraction. It was then washed thoroughly with rigor solution and homogenized by the Cole-Palmer LabGen 125 homogenizer for 10 s followed by further 10-s homogenization after a cool down period of 30 s. Contracting myofibrils were immobilized without affecting their ATPase by cross-linking with the water-soluble cross-linker 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide (EDC) (4, 5, 22, 50). Myofibrils (1 mg/ml) were incubated for 20 min at room temperature with 20 mM EDC in Ca-rigor solution. The reaction was stopped by adding 20 mM DTT. The pH of the solution (7.5) remained unchanged throughout the 20-min reaction. The absence of shortening was verified by labeling the myofibrils with 10 nM rhodamine-phalloidin and observing contraction in a TIRF microscope.

Myosin light chain 1 labeling and exchange into myofibrils.

Recombinant myosin light chain 1 (LC1) was purchased from Prospec (ProSpec-Tany TechnoGene, Rehovot, Israel). It was labeled at position 182 with five molar excess of dye (SeTau-647-maleimide) overnight in ice. Labeled LC1 was purified by passing through a Sephadex G50 LP column to eliminate the free dye. LC1 (and not RLC) was chosen as the site of labeling to ensure that the mutation site was located on a different polypeptide chain so as not to cause any disturbance to the mutation. Freshly prepared myofibrils (1 mg/ml) were exchanged with an exchange solution as described previously (32) with 10 nM SeTau-LC1 under mild conditions (20 min at 30°C; see Ref. 51). The exchange was deliberately made highly inefficient so that only a small fraction of native LC1s were fluorescent. Myosin exchanged with fluorescent LC1 and bound to myofibrils has high anisotropy, in contrast to free LC1, i.e., it is well immobilized by myofibril even though it is bound at one site only.

Choice of the dye.

The fluorophore of choice was SeTau-647. It is excited in the red and hence bypasses most contributions of autofluorescence (30). SeTau is also very resistant to photobleaching (the initial rate of photobleaching was 2.4/s) because of nanoencapsulation of the squaraine moiety of the dye chromophore system in a mixed aliphatic-aromatic macrocycle. SeTau has a large Stokes shift (44 nm), high extinction coefficient, and quantum yield (0.65). Its overall fluorescence intensity was 4.2 times larger than fluorescence intensity of Alexa647.

Data collection and statistical analysis.

The fluorescence was measured by a PicoQuant MT 200 inverse time-resolved fluorescence instrument coupled to an Olympus IX 71 microscope. Before each experiment, fluorescence of an isotropic solution of a long fluorescence lifetime dye (i.e., with 0 anisotropy, such as rhodamine 700) was measured to make sure that the emitted light was split equally by a birefringent prism into the parallel and perpendicular The 640-nm excitation beam was focused by an Olympus ×60, 1.2 numerical aperture water immersion objective to the diffraction limit on the overlap band of a myofibril. For each overlap band, the fluorescence intensity was measured 20,000 times over 20 s. The normalized ratio of the difference between parallel and perpendicular channels is known as polarization of fluorescence (PF). PF is known to be a sensitive indicator of the orientation of fluorophore's transition dipole moment (12, 23, 24, 40, 43, 48, 49). A histogram of PFs, i.e., a plot of PF vs. frequency with which it occurs over 20 s, was constructed from these 20,000 measurements. Myofibrils (∼20–30) were examined for each physiological state (i.e., rigor, contraction, or relaxation), location (i.e., LV or RV), and state of mutation (i.e., WT or MUT) of a ventricle.

The differences between full width at half-maximum (FWHM) values of PF of different populations of ventricles are statistically interpretable only when the signal from each half-sarcomere is similar (38). This is because the relative value of FWHM of a strong random signal is small (relative to a signal), and FWHM of a weak signal is relatively large. To make sure that a signal was equal, the power was adjusted within the 1- to 2-μW range. Comparisons between groups were performed using an unpaired Student's t-test (Sigma Plot 11; Systat Software, San Jose, CA) or Origin version 8.6 (Northampton, MA). The significance was defined as P < 0.05. SigmaPlot 11 was used to compute histograms. Origin was used to compute autocorrelation functions (ACFs).

The number of observed myosin molecules.

The detection volume (DV) was estimated by measuring the width of axial and lateral dimensions of an image of 20-nm fluorescent beads. It was 1.7 μm³ and contained ∼3 × 10⁵ XBs. The number of observed (fluorescent) XBs in this volume was reduced by four orders of magnitude (see below). Data are expressed as means of n experiments ± SD. A calibration curve that plotted the number of SeTau molecules in the DV vs. fluorescence intensity was constructed. The number of molecules in the DV was estimated using fluorescence correlation spectroscopy. The ACF of fluctuations of fluorescence (because of free SeTau entering and leaving the DV) at delay time 0 is equal to the inverse of the number of molecules contributing to the fluctuations (14, 15, 35). Six correlation functions were obtained from solutions of the fluorophore in the range 1.29–77.3 nM. Extrapolating the plot to one molecule revealed that a single molecule of SeTau illuminated with 0.2 μW of laser power gave 225 counts/s (38). We used this fact to estimate the number of molecules contributing to the signal as 20–40. However, it should be emphasized that, as long as the number of XBs is mesoscopic (i.e., when the number of observed molecules is so small that fluorescence is affected by fluctuations around the average; see Ref. 44), the exact number does not matter, i.e., 20 molecules should give the same result as 40 molecules, etc. The precision of measurement is approximately equal to 1/$\sqrt{\text{the}\hspace{0.17em}\text{number}\hspace{0.17em}\text{of}\hspace{0.17em}\text{fluctuations}}$. To measure with 1% precision we must analyze 10,000 fluctuations. The characteristic lifetime of one fluctuation is of the order of 1 ms (26), and 10,000 fluctuations occur in 10 s. We collected the data over 20 s.

Stopped-flow measurements.

Three Tg-WT and seven Tg-K104E mouse hearts were used for mouse cardiac myosin preparation (see above). Myosins at 0.25 μM were mixed with 0.25 μM pyrene-labeled F-actin (stabilized by 0.25 μM phalloidin) in rigor buffer containing 0.4 M KCl, 1 mM DTT, and 10 mM MOPS, pH 7.0. The complexes were mixed in a 1:1 ratio (vol/vol) with varying concentrations of MgATP (10–80 μM) dissolved in the same buffer, and the dissociation of Tg-K104E vs. Tg-WT myosin from F-actin was observed by monitoring the time course of the change in pyrene fluorescence. Measurements were performed at 21°C using a BioLogic (Claix, France) model SFM-20 stopped-flow instrument outfitted with a Berger ball mixer and an FC-20 observation cuvette. The data were collected and digitized using a JASCO 6500 Fluorometer. The estimated dead time was 8.2 ms. The pyrene-F-actin was excited at 347 nm, and emission was monitored at 404 nm using monochromators set to 20-nm bandwidths. Typically, 8–14 stopped-flow records were averaged and fit to a single exponential equation to obtain the rate of a given MgATP concentration. A plot of the observed myosin dissociation rates as a function of [MgATP] was linear, and the slope corresponded to the rate constant expressed in molar per second.

RESULTS

The degree of order (distribution of XB orientations).

An informative way to characterize spatial distribution of XBs is to construct a histogram of polarization values vs. the number of times that a given polarization (i.e., orientation) occurs during a 20-s experiment. Figure 1 is a typical example of histograms of contracting Tg-WT (left) and contracting MUT myofibrils (right). The graphs are Gaussian. The regular residuals are shown (Fig. 1). Goodness of fit to an ideal Gaussian was assessed by adjusted coefficient of determination, AR². It is a better measure of goodness of fit than χ² (typically employed to assess nonlinear curve fitting) because it takes into account scaling of the dependent variable. The closer AR² is to the value of one, the better the fit. Table 1 lists average AR² of 25 experiments of contracting WT and 31 of contracting MUT muscles.

Fig. 1.

Random examples of the probability distribution of orientations of cross-bridges (XBs) of cardiac myofibrils of contracting wild-type (WT) and mutant (MUT) ventricles. Gray line is the best fit to a 3-parameter Gaussian. Graphs on the bottom show regular residuals of the counts. Left: WT muscle, FWHM = 0.464, adjusted coefficient of determination (AR²) = 0.953, mean intensity = 6.3 photons/ms, mean polarization = −0.444. Right: MUT muscle, FWHM = 0.380, AR² = 0.947, mean intensity = 6 photons/ms, mean polarization = −0.156.

Table 1.

Comparison of FWHM's of 25 probability distributions of Tg-WT and 31 experiments of Tg-K104E of contracting myofibrils

Contracting Muscle	FWHM	Intensity, counts/ms	Mean Polarization	AR2
Tg-WT	0.506 ± 0.129*	5.83 ± 2.011	−0.352 ± 0.153*	0.897 ± 0.138
Tg-K104E	0.449 ± 0.046*	4.76 ± 1.112	0.148 ± 0.022*	0.886 ± 0.040

Errors are SDs of 28 experiments.

FWHM, full width at half-maximum; Tg, transgenic; WT, wild type; AR², adjusted coefficient of determination.

^*Statistically significant difference.

Table 1 compares FWHMs of probability distributions of contracting Tg-WT and Tg-K104E (MUT) myofibrils. Detailed results of 25 experiments of contracting Tg-WT are shown in Table 2. Detailed results of 31 experiments of Tg-K104E are shown in Table 3.

Table 2.

Detailed results of probability distributions of contracting Tg-WT myofibrils

Experiment No.	FWHM	TI	Mean Orientation	Adjusted R2
1	0.464	6.39	−0.44	0.953
2	0.35	6.98	−0.627	0.978
3	0.378	8.834	−0.652	0.971
4	0.48	5.918	−0.393	0.972
5	0.821	6.017	−0.361	0.902
6	0.356	10.98	−0.369	0.985
7	0.584	5.86	−0.183	0.953
8	0.76	1.92	−0.319	0.536
9	0.635	4.08	−0.3445	0.909
10	0.511	4.87	−0.444	0.968
11	0.489	6.164	−0.4006	0.955
12	0.463	5.47	−0.291	0.951
13	0.597	3.11	−0.375	0.761
14	0.4312	6.631	−0.4407	0.951
15	0.52038	4.233	−0.291	0.916
16	0.448	5.56	−0.3044	0.946
17	0.4719	7.6	−0.3203	0.965
18	0.491	4.42	−0.397	0.935
19	0.525	4.138	−0.2306	0.874
20	0.46061	4.337	−0.0175	0.851
21	0.656	5.12	−0.012	0.855
22	0.613	4.582	−0.6209	0.411
23	0.489	7.47	−0.4001	0.965
24	0.449	9.66	−0.3359	0.975
25	0.2088	5.58	−0.232	0.976
Average	0.50608	5.837	−0.35206	0.89656
SD	0.12932	2.0114	0.153920258	0.138636

TI, total intensity.

Table 3.

Detailed results of probability distributions of contracting Tg-K104E myofibrils

Experiment No.	FWHM	TI	Mean Orientation	Adjusted R2
1	0.456	4.56	−0.131	0.887
2	0.47001	4.15	−0.167	0.881
3	0.443	5.24	−0.117	0.887
4	0.4332	4.94	−0.1455	0.909
5	0.403	5.321	−0.1553	0.91
6	0.453	4.684	−0.1466	0.905
7	0.392	6.15	−0.123	0.943
8	0.394	5.97	−0.1502	0.924
9	0.38	6.225	−0.156	0.947
10	0.473	4.06	−0.1118	0.853
11	0.423	5.29	−0.1133	0.899
12	0.403	5.388	−0.156	0.921
13	0.497	3.834	−0.1042	0.851
14	0.47	4.21	−0.1255	0.874
15	0.466	4.008	−0.1139	0.846
16	0.48859	4.136	−0.157	0.854
17	0.498	4.051	−0.222	0.897
18	0.554	2.95	−0.177	0.786
19	0.4418	5.108	−0.1859	0.899
20	0.336	8.309	−0.173	0.974
21	0.455	4.54	−0.1849	0.899
22	0.5511	3.28	−0.173	0.807
23	0.476	4.29	−0.1008	0.85
24	0.479	3.81	−0.1618	0.862
25	0.444	4.618	−0.157	0.891
26	0.428	5.232	−0.172	0.931
27	0.412	5.231	−0.184	0.907
28	0.473	4.09	−0.127	0.873
29	0.487	3.77	−0.114	0.833
30	0.456	4.34	−0.1609	0.874
31	0.408	5.139	−0.137	0.893
Average	0.4498	4.765	−0.148348	0.8860323
SD	0.04684	1.112	0.0299128	0.0401501

The t-test revealed that the 0.057 difference of FWHM was statistically significant (t = 2.436, P = 0.018) with 54 degrees of freedom. The 95% confidence interval of this difference is from 0.010 to 0.103. The 1.073 difference of intensities was statistically insignificant (t = 2.532, P = 0.014) with 54 degrees of freedom. The 95% confidence interval of this difference is from 0.223 to 1.922.

The differences in FWHM and polarization values were significant, but the differences in intensities were insignificant. This is important because, as already mentioned in materials and methods, the stochastic nature of a Gaussian signal causes a relative FWHM to depend on the strength of the signal. Weak signal results in a large FWHM, and strong signal results in small FWHM. Thus, when comparing FWHMs of two populations, it is crucial that the difference in photon counts between the two populations be statistically insignificant (38). The matter is more fully discussed in the discussion. The −0.498 difference of mean polarization was statistically significant (t = 17.744, P < 0.001) with 54 degrees of freedom. The 95% confidence interval of this difference is from −0.554 to −0.441.

Figure 2 is a typical example of histograms of rigor Tg-WT (left) and rigor MUT myofibrils (right). As before, the graphs are Gaussian. The regular residuals are shown in Fig. 2.

Fig. 2.

Random examples of the probability distribution of orientations of XBs of cardiac myofibrils of rigor transgenic (Tg)-WT and MUT ventricles. Gray line is the best fit to a 3-parameter Gaussian. Graphs on the bottom show regular residuals of the counts. Left: WT muscle, FWHM = 0.469, AR² = 0.932, mean intensity = 4.7 photons/ms, mean polarization = −0.269. Right: mutated muscle, FWHM = 0.563, AR² = 0.942, mean intensity = 6.817 photons/ms, mean polarization = −0.223.

Table 4 shows a comparison of FWHMs of 25 probability distributions of rigor Tg-WT and 34 experiments of Tg-K104E.

Table 4.

Comparison of FWHM's of 28 probability distributions of Tg-WT and Tg-K104E of rigor myofibrils

Rigor Muscle	FWHM	Intensity, counts/min	Mean Polarization	AR2
Tg-WT	0.579 ± 0.223*	5.68 ± 1.72	−0.226 ± 0.290	0.885 ± 0.101
Tg-K104E	0.484 ± 0.083*	5.53 ± 0.93	−0.229 ± 0.085	0.922 ± 0.032

Values are means ± SD. Errors are SDs.

^*Statistically significant difference.

The t-test revealed that the 0.094 difference of FWHM was statistically significant (t = 2.253, P = 0.028) with 55 degrees of freedom. The 95% confidence interval of this difference is from 0.010 to 0.178. The 0.158 difference of intensities is statistically insignificant (t = 0.4477, P = 0.656) with 55 degrees of freedom. The 95% confidence interval of this difference is from −0.549 to 0.865. The importance of this difference being insignificant is addressed in the discussion. The 0.003 difference of mean polarization is statistically insignificant (t = 0.057, P = 0.954) with 55 degrees of freedom. The 95% confidence interval of this difference is from −0.10244 to 0.10844. Statistically significant differences are shown.

A relevant negative control experiment is to measure distribution in relaxation because here neither WT nor K104E XBs interact with the thin filaments. We therefore expect no differences. Figure 3 is a typical example of histograms of relaxed Tg-WT (left) and relaxed MUT myofibrils (right). As before, the graphs are Gaussian. The regular residuals are shown in Fig. 3.

Fig. 3.

Random examples of the probability distribution of orientations of XBs of relaxed cardiac myofibrils of rigor Tg-WT and MUT ventricles. Gray line is the best fit to a 3-parameter Gaussian. Graphs on the bottom show regular residuals of the counts. Left: WT muscle, FWHM = 0.429, AR² = 0.927, mean intensity = 5.0 photons/ms, mean polarization = −0.074. Right: mutated muscle, FWHM = 0.467, mean intensity = 4.1 photons/ms, mean polarization = −0.100.

Table 5 is a comparison of FWHMs of 28 probability distributions of relaxed Tg-WT and Tg-K104E.

Table 5.

Comparison of FWHM's of 28 probability distributions of Tg-WT and Tg-K104E of relaxed myofibrils

Relaxed Muscle	FWHM	Intensity, counts/min	Mean Polarization	AR2
Tg-WT	0.438 ± 0.037	4.99 ± 0.770	−0.144 ± 0.085*	0.910 ± 0.054
Tg-K104E	0.440 ± 0.043	4.72 ± 0.76	−0.096 ± 0.022*	0.901 ± 0.056

Errors are SDs of 28 experiments.

^*Statistically significant difference.

The t-test revealed that the −0.0025 difference of FWHM was statistically insignificant (t = 0.251, P = 0.802) with 65 degrees of freedom. The 95% confidence interval of this difference is from −0.0223712 to 0.0173712. The −0.271 difference of intensities was statistically insignificant (t = −1.406, P = 0.165 with 63 degrees of freedom). The 95% confidence interval of this difference is from −0.655 to 0.114. The −0.0478000 difference of mean polarization was statistically significant (t = 2.893, P = 0.052 with 55 degrees of freedom). The 95% confidence interval of this difference is from −0.0807955 to −0.0148045.

XB kinetics.

Because the number of observed molecules was small, we were able to measure fluctuations in equilibrium values of PF. These fluctuations contain information about the rate constants characterizing the enzymatic cycle of actin-myosin interaction. An ACF of PF fluctuations characterizes these rate constants. The shape of the ACF depends on the model assumed for the chemical reaction that drives the cycling of XBs. We use combination of models of Lymn and Taylor (34) and of Coureux et al. (10) that includes transitions between three fundamental states as illustrated in Fig. 4 where a XB, actin, ATP, and ADP are denoted by M, A, T, and D, respectively.

Fig. 4.

Conventional model of muscle contraction. In vitro measurements (see materials and methods) have shown that steady-state anisotropy associated with different cross-bridge states are 0 for MDP and MT (dissociated XBs) states, intermediate for the AMDP state, and maximum for AM and AM* states (data not shown). M, XB; A, actin; T, ATP; D, ADP.

The enzymatic (and the mechanical cycle coupled to it) begins with a state MT where M has already dissociated from thin filament by binding T and T is hydrolyzed to D and P to assume the MDP state (34). The products of hydrolysis remain bound to M. In this state cleft between upper and lower subdomains of a 50-kDa domain is partially closed, and the lever arm is in the up position (10). M and MT are free to rotate in the myofilament space and have the lowest steady-state anisotropy (and PF). MDP binds actin with a rate constant k₁ forming a weakly bound, prepower stroke state, AMDP. It is partially immobilized. In this state the cleft between the upper and lower subdomains of a 50-kDa domain is completely closed, and the lever arm is less up than previously (10). Next, P dissociates from AMDP forming a strongly bound rigor complex AMD. It is assumed that this transition is very rapid. The transition from AMD to AM state is a power stroke that occurs with the rate constant k₂. In AM state the cleft is completely closed and the lever arm is in the down position. The anisotropy is high. Finally, AM isomerizes to a “rigor-like” state with a rate constant k₃ (10). In this state the cleft is opened and the lever arm becomes uncoupled from actin. Anisotropy is rigor-like. The true relaxation is the transition from AM* to MT state characterized by the rate k_obs.

A representative experimental ACF is plotted in Fig. 5. The red line shows the best nonlinear fit to theoretically predicted expression of the ACF of a system of in Fig. 4 (an analytical expression of ACF is shown in Ref. 36). The kinetic constants were reported earlier by Huang et al. (25) and are reproduced as follows: k₁ = 205 ± 93.7/s, k₂ = 0.617 ± 0.496/s, and k₃ = 0.689 ± 0.499/s for Tg-WT myofibrils and k₁ = 779 ± 572.1/s, k₂ = 1.162 ± 1.198/s, and k₃ = 0.300 ± 0.242/s for Tg-K104E myofibrils.

Fig. 5.

A representative trace of a normalized autocorrelation function (ACF) of polarization of fluorescence of contracting myofibril prepared from the left ventricle of Tg-WT mouse. Symbols are experimental data, and a gray line is the fit to equation shown in Ref. 36. The results of each experiment are presented in Table 4. The fact that ACF decays to a value >0 is due to the fact that mean polarization was nonzero.

The t-test revealed that the −574 difference of k₁ was statistically significant (t = −4.764, P < 0.001) with 50 degrees of freedom. The −0.545 difference of k₂ was statistically significant (t = −2.091, P = 0.041) with 53 degrees of freedom. The +0.389 difference of k₃ was statistically significant (t = 3.809,P = 0.001) with 53 degrees of freedom.

The detailed results of kinetics experiments on WT and mutated ventricles are shown in Table 6.

Table 6.

Detailed results of 23 kinetics experiments on WT ventricles and 29 experiments on mutated ventricles

k1 WT	k1 MUT	k2 WT	k2 MUT	k3 WT	k3 MUT
244.2160	529.4700	0.7680	0.3310	0.8530	0.1590
189.4840	471.8200	0.1140	0.0450	0.1200	0.0281
2.1650	343.5200	0.2850	3.4400	0.8770	0.2700
1.8450	347.6000	0.4020	0.0440	0.5240	0.0330
138.5100	368.3400	0.6970	0.5300	1.8400	0.3100
319.8440	1,535.0060	0.4850	0.3330	0.9630	0.1810
218.8400	376.1900	1.5280	3.3350	1.3590	0.9800
359.5600	1,644.4000	0.4440	1.0400	0.2500	0.4850
226.4500	1,442.2400	0.9170	1.2680	0.5600	0.5970
284.5820	416.8900	0.7800	0.6290	1.2700	0.0580
219.4140	337.1720	0.0530	0.0320	0.0390	0.0150
208.1330	1,477.1570	0.6300	2.0100	0.6690	0.6200
x	355.6100	0.7370	0.1670	0.5590	0.0400
200.7300	422.7700	0.2500	2.1570	0.3140	0.3320
238.0140	308.4370	0.0370	0.0710	0.0290	0.0230
263.8200	1,519.7680	0.3700	0.9530	0.3410	0.4600
213.2600	306.0660	0.1660	0.4080	0.1730	0.2340
302.3300	1,444.8200	0.8620	4.7900	1.2080	0.5600
226.8060	340.6200	0.5610	0.2700	0.4030	0.1620
−0.1250	402.2800		0.3160	0.1246	0.2180
247.4680	x	2.3700	2.2700	x	0.7900
x	450.8900	0.6400	2.3900	0.5170	0.3930
224.1660	419.7400	0.6730	2.3000	1.3670	0.3120
232.5190	304.6900	0.5211	0.0548	0.9610	0.0230
174.0300	1,690.4300	0.5260	0.8750	1.2250	0.3900
	1,036.8661		0.9740		0.5240
	1,839.0600		1.4200		0.3330
	397.8100		0.0700		0.0540
	x		2.1760		0.1219
	1,596.7300		0.7430		0.3590
	367.5150		0.6020		0.2360

Values are means ± SD. x, Extreme values of constants that were removed from the original data. Extremes were defined as differing by more than a factor of 5 from the mean.

The mesoscopic method was unable to measure the rate of XB dissociation of Tg-K104E and Tg-WT myosin from thin filaments (k_obs). To measure this rate actin was labeled with pyrene (31). The time course of the change in pyrene fluorescence was monitored as a function of ATP concentrations. Myosins were stoichiometrically mixed with pyrene-F-actin, and the complexes were mixed in a 1:1 volume ratio with increasing concentrations of MgATP (10–80 μM) in a stopped-flow apparatus. An increase in the fluorescence intensity was monitored as a function of time as the myosin heads dissociated from pyrene-F-actin on the addition of MgATP. The representative time-dependent dissociation profiles for Tg-K104E myosins are presented in Fig. 6A. The k_obs, derived from the averaged fluorescence traces and fitted with a single exponential, are presented in Table 7. The results revealed significantly faster k_obs in Tg-K104E vs. Tg-WT myosin for 10 and 25 μM MgATP and no differences between K104E and WT at higher ATP concentrations. Linear regression analysis of the plot of k_obs vs. increasing [MgATP] yielded the effective second-order ATP-binding rate for the K104E equal to 0.68 ± 0.01 μM⁻¹/s compared with 0.74 ± 0.02 μM⁻¹s for Tg-WT (Fig. 6B) showing a small but significant (P < 0.05) change in the slope indicating a smaller MgATP binding rate for the mutant.

Fig. 6.

Fast kinetics of interaction between Tg-K104E and Tg-WT myosins with pyrene-labeled F-actin. The myosin-pyrene-actin complex was mixed with various MgATP concentrations (range from 10 to 80 μM), and the recovery of pyrene fluorescence was monitored on the dissociation of actomyosin complex. A: representative traces of a real-time increase of pyrene fluorescence within 1 s after MgATP was added to the Tg-WT-F-actin system. B: observed dissociation rates (k_obs)-[MgATP] dependence and the effective second-order MgATP-binding rates for Tg-K104E vs. Tg-WT myosins. The average values of k_obs ± SE for each MgATP concentration are presented in Table 7.

Table 7.

Observed dissociation rates (s⁻¹) for Tg-WT and Tg-K104E myosins

	Myosin/[ATP], μM
	10	25	40	60	80
Tg-WT	3.95 ± 0.081	11.75 ± 0.69	26.31 ± 0.41	36.26 ± 1.24	60.56 ± 2.84
n	11	11	11	11	11
Tg-K104E	6.71 ± 0.49*	18.08 ± 0.45*	27.26 ± 0.41	39.08 ± 0.89	55.81 ± 1.44
n	22	22	22	22	22

Errors are SEs of n sets of experiments with each set including at least 5 independent measurements. Concentrations refer to the amount of [ATP].

^*P < 0.01.

DISCUSSION

We studied the effect of the K104E mutation in RLC on the degree of order of ventricular myosin during steady-state contraction, relaxation and rigor, and on the XB kinetics during contraction of left ventricular myofibrils. Autofluorescence and photobleaching were minimized by labeling the myosin lever arm with a relatively long-lived red-emitting dye. Inhomogenities were minimized by focusing the exciting laser light on a single half-sarcomere. The dye was immobilized during critical transient states of the contraction cycle, in spite of the fact that it was attached to LC1 at one point only, rather than two points, which is more stable (9). By using the mesoscopic approach, we were able to measure the average kinetics of ∼20 XBs.

The degree of order.

As mentioned before, comparison of the probability distributions of orientations of Tg-WT and Tg-K104E XBs can be misleading unless the means of distributions are the same (38). The relative FWHM of distribution [for normal distribution FWHM = 2$\sqrt{2\text{In}2}$ = 2.355 SD] is sharply dependent on the strength of the signal. A weak Gaussian signal is associated with a broad distribution, and a strong Gaussian signal is associated with a narrow distribution. The same is true for any ratio of two Gaussian signals (like PF) (38). Therefore, meaningful comparison of FWHMs (or SDs) of the polarization of the two Gaussian signals requires that they have nearly equal mean. In practice, we ensure this by keeping the average photon count in each series of experiments similar (by adjusting the power of the illuminating laser). Because the difference in the intensities was statistically insignificant, we could conclude that, during contraction and rigor, the Tg-WT XBs were distributed more widely than mutated Tg-XBs. The fact that the spatial distribution of contracting XBs was significantly narrower in the Tg-K104E myofibrils was not surprising since mutated muscle was found to develop less tension than WT muscle (25), implying that K104E-XBs cannot reach as many actin-binding sites as WT-XBs. The observation of XB order in contraction is consistent with earlier work (7). The difference in polarization values of XBs of relaxed K104E and WT myofibrils was significant, perhaps because some myosin-actin interaction occurs at relatively low ionic strength used here (8). It was surprising to find that FWHM values for both types of myofibrils were not wider in relaxation compared with rigor.

Kinetics of contracting myofibrils.

The analysis of the ACF leads to two immediate conclusions about the mode of action of XBs. First, the action of XBs is not synchronized. This is because the ACF obtained from XBs within a single half-sarcomere were not periodic. If they were periodic, the XBs would have to be synchronized because ACF of a periodic signal is periodic (6). Second, although cross-linking prevented shortening, it did not prevent XBs from executing the power stroke. The present work shows that XBs rotate and are able to reach the neighboring target zones. Not only enzymatic but also mechanical cycles go on, in spite of the cross-linking (22).

Kinetic results show that the K104E mutation exerts significant effects on the interaction between actin and myosin. It increases the rates of XB binding, execution of the power stroke, and XB dissociation from the thin filaments. In particular, the rate of XB dissociation from the thin filaments is increased. This suggests that a mutated ventricle is prone to relaxation abnormalities, ultimately leading to diastolic dysfunction in patients carrying the mutation. The molecular effects of the mutation are shown schematically in Fig. 7. The blue cones (that indicate the cone of angles within which the transition dipole can rotate) suggest that the degree of disorder of XBs in WT ventricle (top) is larger than in K104E XBs (bottom). This implies that K104E-XBs cannot reach as many actin-binding sites as WT-XBs, i.e., we could predict that, in vivo, the mutated ventricles would develop less tension compared with WT. This prediction was confirmed by Huang et al. (25).

Fig. 7.

Schematic representation of XBs in contracting WT (top) and K104E (bottom) ventricles. WT XBs are more disorganized than K104E XBs [they rotate within larger cone of angles (blue)]. K104E XBs dissociate faster than WT (there are less XBs on bottom). Gray spheres are the fluorescent LC1s. Even though they can interact with actin (3), for simplicity it is drawn here separate from actin. Red ellipsoid represents the 50 + 20-kDa NH₂-terminal domains of the myosin head. Myosin thick filament is shown in light blue, actin filament in yellow, and the COOH-terminal domain of XB (lever arm) in green.

The difference in XB distribution and kinetics between K104E and WT may constitute a molecular hallmark of FHC. Ventricles carrying familiar hypertrophy mutations at different sites also show obvious differences in the kinetics compared with WT muscles (13, 20, 21, 29, 36, 37, 39). Differences are also present between the α- and β-cardiac myosins (33).

In conclusion, we report here that the K104E mutation significantly alters the degree of order and the kinetics of single myosin XBs carrying the mutated RLC. The mutation-imposed molecular changes monitored in Tg-K104E myofibrils are followed by physiological changes in heart contractility of transgenic mice. We conclude that the differences in XB distribution and kinetics between the K104E mutant and WT muscles are sufficient to trigger pathological heart remodeling and FHC.

GRANTS

This work was supported by National Institutes of Health Grants R01-AR-048622 (J. Borejdo), R01-HL-108343 (D. Szczesna-Cordary), and R01-HL-090786 (J. Borejdo and D. Szczesna-Cordary) and by American Heart Association Grant 12PRE12030412 (W. Huang).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: D.D., J.N., R.R., W.H., K.M., R.F., and D.S.-C. performed experiments; D.D., J.N., R.R., K.M., and J.B. analyzed data; D.D. and J.N. prepared figures; R.R. and J.B. interpreted results of experiments; R.R., I.G., and J.B. approved final version of manuscript; R.F. edited and revised manuscript; I.G., D.S.-C., and J.B. conception and design of research; J.B. drafted manuscript.

Glossary

ACF

Autocorrelation function

DV

Detection volume

FHC

Familial hypertrophic cardiomyopathy

FCS

Fluorescence correlation spectroscopy

FWHM

Full width at half-maximum

LC1

Myosin light chain 1

MF

Cardiac myofibrils

PF

Polarization of fluorescence

RLC

Regulatory light chain of myosin

WT

Wild type

XB

Cross bridge