Sarcomere, troponin, and myosin X-ray diffraction signals can be resolved in single cardiomyocytes

Abstract

Cardiac function relies on the autonomous molecular contraction mechanisms in the ventricular wall. Contraction is driven by ordered motor proteins acting in parallel to generate a macroscopic force. The averaged structure can be investigated by diffraction from model tissues such as trabecular and papillary cardiac muscle using collimated synchrotron beams, offering high resolution in reciprocal space. In the ventricular wall, however, the muscle tissue is compartmentalized into smaller branched cardiomyocytes, with a higher degree of disorder. We show that X-ray diffraction is now also capable of resolving the structural organization of actomyosin in single isolated cardiomyocytes of the ventricular wall. In addition to the hexagonal arrangement of thick and thin filaments, the diffraction signal of the hydrated and fixated cardiomyocytes was sufficient to reveal the myosin motor repeat (M3), the troponin complex repeat (Tn), and the sarcomere length. The sarcomere length signal comprised up to 13 diffraction orders, which were used to compute the sarcomere density profile based on Fourier synthesis. The Tn and M3 spacings were found in the same range as previously reported for other muscle types. The approach opens up a pathway to record the structural dynamics of living cells during the contraction cycle, toward a more complete understanding of cardiac muscle function.

Figure 1

(a) Sketched myofibril, the contraction organelle of cardiomyocytes, which consists of a series of (b) sarcomeres of length $1.7-2.5\mu$m, in which thin and thick filaments are suspended in a hexagonal lattice giving rise to the $d_{1,0}$ and $d_{1,1}$ peak in Fig. 3. The types of filaments can slide relative to each other. The actin-based thin filaments are connected to the neighboring sarcomere at the Z-line by α-actinin (37). Between two Z-lines, the myosin-containing thick filaments are more loosely suspended from the M-line. (c) Along the thin filament, the actin helix has a $35.75$ nm pitch equal to that of the regulatory tropomyosin complex surrounding it but different from the spacing between troponin (Tn) molecules of $38.7$ nm (also see d). The thick filament holds myosin motors in a quasi-helical arrangement with a $42.9$ nm repeat. Every half-filament has 49 diffraction layers $\approx 14.3$ nm apart, with three myosin motors causing the M3 reflection in Fig. 3c (cf. (7)). (d) Thin filament components and myosin motor shown in the on and off states, the latter of which is called the interacting head motif (ihm) (38). In the C-zone from diffraction layer 7 to 31, the off state is suspected to be stabilized by the myosin binding protein C (MyBP-C), which is positioned on the myosin tail. The P and D zones do not contain the MyBP-C (29). To see this figure in color, go online.

Figure 2

Sample environment and data acquisition. (a) Sketch of the steel-frame-supported $1\mu$m polypropylene (pp) foil windows, coated with laminin for cell adhesion. Two frames are combined to form $\approx 0.5$-mm-thick wet compartments held by a three-dimensional-printed holder (blue structure in the photograph). (b) Beamline setup at ID02 (ESRF) with X-ray undulator source (u), a Si-111 channel-cut monochromator (mm), mirrors (mr), and slits (sl) removing stray scattering before the sample (s). The diffraction patterns are recorded with an Eiger 4M photon counting pixel detector (d) at a distance of 3 or 31 m. (c) 40× phase-contrast microscopy mosaic showing the $z=3$ m scan in cyan and the $z=31$ m scan with illumination time ($\tau =0.1$ s) in magenta. (d) 40 × 40 px² sSAXS map of dark-field X-ray intensities in a radial $q_r\in \left[0.1,0.2\right]$ range, with a $50\mu$m step size ($\tau =0.5$ s). (e) Diffraction patterns are recorded at pixel positions marked in the map (d) by the open magenta squares, exhibiting equatorial and meridional reflections. The corresponding phase-contrast micrographs of the corresponding cells (numbered 1–5 and 8) are also shown. Scale bars, $30\mu$m. To see this figure in color, go online.

Figure 3

Cellular diffraction signals. (a) Diffraction pattern of single ventricular cardiomyocyte recorded for $\tau =0.5$ s at a sample detector distance $z=3$ m with rectangles showing summation areas of meridian profiles depicted in (c) and equatorial profiles depicted in (d) (colors indicate opposing sides in a). (b) Phase-contrast microscopy of the corresponding cell (cell number 1) showing the position of the nucleus (white triangle) (scale bar, $50\mu$m). (c) Background-corrected Meridian showing the M3 peak and the Tn reflection. A pronounced split is observed for the M3 reflection. (d) Equatorial profile with the $d_{1,0}$ and $d_{1,1}$ reflections, indicative of the actomyosin lattice spacing (see Fig. 1). To see this figure in color, go online.

Figure 4

Sarcomere reflections and density profile. (a) Diffraction pattern recorded at $z=31$ m with an illumination time $\tau =0.1$ s exhibiting sarcomeric reflections of orders $n=2$ to 14 (highlighted area) shown as a one-dimensional profile in (b). The positions of the peaks were fitted to Gaussians and plotted against n in (c). These data were fitted by error-weighted linear regression to yield an average $SL=\left(1816\pm 2\right)$ nm. (d) Relative peak intensity $I_{\mathrm{X}-\text{ray}}$ from (b) (linear background subtraction) times $n^2$ (purple) with the real part of a Fourier transform of an electron microscopy profile ${\rho }_e$ included as a reference (green, taken from (43); for details, see Fig. S4). The comparison helps to find the (unknown) signs for the Fourier synthesis. (e) Resulting sarcomere density profile $\rho \left(z\right)$. (f) The dark-field intensity of cell 1, with dark-field mask given by the cake profile in (a), measured with a $10\mu$m step size at $z=31$ m. (g) Map of the $SL$ of the cell. (h) Error ${\sigma }_{SL}$ of the $SL$. (i) Histogram of obtained $SL$ values. To see this figure in color, go online.

Figure 5

Parameters of equatorial reflections. The data presented in Fig. 3d reflect the hexagonal ordering of the myofilaments orthogonal to the filament axis (see Fig. 1a). The parameters determined by least-square fitting are plotted against each other to identify correlations and dependencies. (a–c) Two-dimensional histograms of parameter combinations with correlation coefficients $r>0.8$, including (a) $I_{1,0}$ versus $d_{1,0}$, (b) $I_{1,1}$ versus $I_{1,0}$, and (c) both relative intensities $I_{1,1}$ and $I_{1,0}$ showing a similar decay with low $d_{1,0}$ spacing. To see this figure in color, go online.

Figure 6

Tn. (a) Sketch of the on and off states of the actin-based thin filament with tropomyosin (yellow) rotated by a conformational change in Tn to a more disordered state. (b and c) Intensity of the Tn reflection $I_{\text{Tn}}$, a measure of the Tn order, plotted against (b) the lattice parameter $d_{1,0}$ and (c) the equatorial reflection intensity $I_{1,0}$, each showing a positive correlation with $I_{\text{Tn}}$. (d) Tn spacing $d_{\text{Tn}}$ plotted against $d_{1,0}$. To see this figure in color, go online.

Figure 7

M3 peak analysis. (a) The sum of both M3 reflections of each diffraction pattern (1–12, shifted for visibility) fitted by model Eq. 2 (black). (b) Illustration of the thick filament in a relaxed state (top) and at peak force (bottom), showing the ordered myosin heads that contribute to scattering (dark brown). They are back-folded (short) or bound to the thin filament (long). Disordered relaxed myosin heads (light brown) do not contribute to the M3 signal. Each filament forms two interfering diffracting lattices with a center to center interference distance $ID$. The diffraction pattern of this structure is a superposition of the two-structure diffraction from both lattices and the lattice diffraction pattern itself, as illustrated in (c). (d) Myosin motor spacing $d_{\mathrm{M}3}$ is weakly negatively correlated with the filament lattice spacing $d_{1,0}$, whereas (e) $ID$ and (f) the intensity $I_{\mathrm{M}3}$ are uncorrelated $d_{1,0}$. To see this figure in color, go online.

Figure 8

Additional diffraction signal tentatively attributed to collagen. (a) Diffraction pattern of cell 4 with diffraction maxima away from the equatorial and meridional plane (indicated by arrows). (b) Confocal fluorescence image showing (labeled) collagen in a myocyte of the left ventricular wall of a canine heart; the longitudinal axis is oriented from left to right. Scale bar, $25\mu$m, from (59). (c) Sketch of myofibrils, responsible for the sarcomere equatorial peaks, and the crossed collagen fibrils suspected to cause the collagen reflections based on the fibril’s width and dense packing with periodicity $\left(33\pm 2\right)$ nm (55). The collagen fibrils are at an angle ψ relative to the layer lines of the myofibrils. (d) Radial intensity profile of cell 4 in an area excluding equator and meridian (green), fitted by the sum of a Gaussian and an exponential function revealing a periodic structure with a lattice constant of $34.4\pm 0.2$ nm. (e) Angular profile (ring in a) showing the angular offset ψ from the meridian. (f) The geometric prefactor $f\left(\psi \right)$ of the collagen weaves contribution to Young’s modulus to the combined myofibril structure at a given helix pitch $\left|\psi \right|$ for 10 of the diffraction patterns. (g) Histogram of the absolute angular orientations $\left|\psi \right|$ determined as shown in e) of each side is scattered along the curve and distributed around $\left|\psi \right|={55}^{\circ }$. To see this figure in color, go online.