Kinetics of a single cross-bridge in familial hypertrophic cardiomyopathy heart muscle measured by reverse Kretschmann fluorescence

Abstract

Familial hypertrophic cardiomyopathy (FHC) is a serious heart disease that often leads to a sudden cardiac death of young athletes. It is believed that the alteration of the kinetics of interaction between actin and myosin causes FHC by making the heart to pump blood inefficiently. We set out to check this hypothesis ex vivo. During contraction of heart muscle, a myosin cross-bridge imparts periodic force impulses to actin. The impulses are analyzed by fluorescence correlation spectroscopy (FCS) of fluorescently labeled actin. To minimize observation volume and background fluorescence, we carry out FCS measurements in surface plasmon coupled emission mode in a reverse Kretschmann configuration. Fluorescence is a result of near-field coupling of fluorophores excited in the vicinity of the metal-coated surface of a coverslip with the surface plasmons propagating in the metal. Surface plasmons decouple on opposite sides of the metal film and emit in a directional manner as far-field p-polarized radiation. We show that the rate of changes of orientation is significantly faster in contracting cardiac myofibrils of transgenic mice than wild type. These results are consistent with the fact that mutated heart muscle myosin translates actin faster in in vitro motility assays.

Figure 1

Concept of SPAM microscope. A cardiac myofibril is illuminated from above. (a) In a conventional microscope all light, including scattered (background) light, is able to penetrate the coverslip. Green dots represent fluorophores that are out of the field of excitation. Red dots represent fluorophores that are in the path of direct or scattered excitation light. (d) In RK/SPAM, a sample is placed on a metal-coated coverslip and excited with green light (right). The excitation energy from the excited fluorphore couples to the surface plasmons and radiates through the metal film (red) to the objective as a surface of a cone with a half angle equal to the SPCE angle. Metal can be a thin layer of Al (20 nm thick), or Ag or Au (50 nm thick). The scattered light is unable to penetrate the coverslip and is radiated into free space. (b) and (e) The background rejection by SPAM. 0.5-mM rhodamine 800 added as background obscures the image in ordinary TIRF (b). SPAM in the RK configuration eliminates much of the background contribution (e). Myofibrils (0.1 mg∕mL) were labeled with 100-nM Alexa647-+10-μM unlabeled phalloidin for 5 min at room temperature, then extensively washed with rigor buffer containing 50-mM KCl, 2-mM MgCl₂, 1-mM DTT, 10-mM TRIS pH 7.0. 633-nm excitation, 1.65 NA 100× Olympus objective, sapphire substrate, 1.78 refractive index immersion oil. The bars are 5 μm in (b) and 10 μm in (e). (c) and (f) The back focal plane image of a sample in a microscope in (c) conventional configuration consists of weak outer ring and a diffuse interior with s strong center corresponding to imperfect blockage of exciting light. The BFP image of a sample in a microscope in (f) RK configuration consists of a strong ring corresponding to emission into free space in a cone.

Figure 2

(a) Power entering the objective at various distances from the surface of a coverslip. (b) The SPCE (solid line) and TIRF (broken line) power entering the objective at various polar angles. Curves normalized to power at θ=0 deg.

Figure 3

RK-SPAM FCS experiments using fluorescent spheres diffusing on glass, gold-coated glass, and gold-coated sapphire coverslips. (a) Correlation function of spheres diffusing on glass (red), gold-coated glass (green), and gold-coated sapphire (blue). The average number of spheres in DV was 6, 8, and 22, respectively. (b) The decay of the correlation function is fastest for spheres diffusing on gold-coated sapphire and slowest for glass. The total photon rate was ≈600 Kcounts∕s. The incident power is 150 μW. (Color online only.)

Figure 4

Time traces from the left ventricle on gold-coated glass labeled with 10-nM rhodamine phalloidin+10-μM unlabeled phalloidin. (a) Contracting and (b) rigor muscle. Parallel (I_H) and perpendicular (I_V) intensities are shown in the blue and red, respectively. Insets show histograms of counts during (a) contraction and (b) rigor. Note that the Gaussian curve used to fit the contraction data is asymmetrical. (Color online only.)

Figure 5

Correlation function of Tg-R58Q myofibrils. (a) Contracting myofibrils. (b) Rigor myofibrils. red—autocorrelation function of ch1 (I_H), green—ch2 (I_V), blue—cross-correlation ch1×ch2. (Color online only.)

Figure 6

Pre-exponential constant B from a fit of WT (left) and mutated (right) myofibrils. The error bar is SD. N=5 for R58Q, 4 for WT.