Features of endogenous cardiomyocyte chromatin revealed by super-resolution STED microscopy

Abstract

Despite the extensive knowledge of the functional unit of chromatin-the nucleosome-for which structural information exists at the atomic level, little is known about the endogenous structure of eukaryotic genomes. Chromosomal capture techniques and genome-wide chromatin immunoprecipitation and next generation sequencing have provided complementary insight into global features of chromatin structure, but these methods do not directly measure structural features of the genome in situ. This lack of insight is particularly troublesome in terminally differentiated cells which must reorganize their genomes for large scale gene expression changes in the absence of cell division. For example, cardiomyocytes, which are fully committed and reside in interphase, are capable of massive gene expression changes in response to physiological stimuli, but the global changes in chromatin structure that enable such transcriptional changes are unknown. The present study addressed this problem utilizing super-resolution stimulated emission depletion (STED) microscopy to directly measure chromatin features in mammalian cells. We demonstrate that immunolabeling of histone H3 coupled with STED imaging reveals chromatin domains on a scale of 40-70 nm, several folds better than the resolution of conventional confocal microscopy. An analytical workflow is established to detect changes in chromatin structure following acute stimuli and used to investigate rearrangements in cardiomyocyte genomes following agonists that induce cellular hypertrophy. This approach is readily adaptable to investigation of other nuclear features using a similar antibody-based labeling technique and enables direct measurements of chromatin domain changes in response to physiological stimuli.

Fig. 1

Super-resolution visualization of cardiac chromatin via STED microscopy. Top panels, left to right: confocal imaging of histone H3; STED image of the same nucleus; and zoom image of region indicated by arrow in bottom middle panel. Individual pixels in the STED images are 14–16 nm. Bottom panels, higher magnification images of boxed area in top panels: confocal (false colored green); STED; and overlay. Bar is 1 μm in all images except top right, where it is 50 nm.

Fig. 2

Resolution of STED. Panel A, line scans demonstrate vastly superior resolution obtained with STED as compared to conventional confocal imaging of the same field. Panels iii and iv are zoom images of i and ii, respectively; panel v shows the line scan profile which displays resolution of two distinct foci (red line; distance between STED peaks is 175 nm) in STED image that are not resolved in the conventional confocal (gray line) data. Inset in panels iii and iv shows the area of analysis at higher magnification. Typical resolution, measured at full-width half-maximum (FWHM), with this method is 40–70 nm (see also Supplemental Figs. 1B and C). Using this method, nuclei from HeLa (panel B) and HEK293 cells (panel C) were similarly labeled with histone H3 antibodies and the same field visualized via conventional confocal or STED microscopy. Panel D, serial z stack STED images of histone H3 in myocytes (taken at 0.5 μm intervals along the z axis) demonstrate architecture of chromatin throughout the in situ nucleus. Bar is 1 μm in all images except insets where it is 0.2 μm.

Fig. 3

Localization of RNA POLII and its features as revealed by STED. Panel A, NRVMs were labeled with histone H3 and RNA POLII, simultaneously visualized via STED (red) or conventional confocal (cyan), respectively. Panel B, same as previous panel with the RNA POLII visualized in STED channel (red) and histone H3 in conventional confocal (cyan). RNA POLII displayed diffuse staining throughout the nucleus, with concentrations in the nuclear periphery. This pattern was unique to NRVMs, as neurons displayed a more homogenous distribution of RNA POLII despite a similar global histone H3 pattern (panel C). Bar is 1 μm in all images.

Fig. 4

Global chromatin changes after hypertrophic agonists detected by STED microscopy. NRVMs were treated with isoproterenol (ISO, 1 μM) or vehicle and examined 24 or 48 h later. Panel A, STED images of histone H3 labeling were acquired in each condition and intensity contour analysis performed, examples of which are shown in this panel. Bar is 1 μm. Panel B, ISO induces hypertrophy in NRVMs, as described [18]. Cells are labeled with alpha-actinin and visualized by confocal microscopy at either 24 (top panels) or 48 (bottom panels) hours after ISO. Bar is 50 μm. Histogram displays quantitative data on cell size changes (SEM displayed). Panel C, STED images from histone H3 labeled vehicle or ISO-treated cells (24 or 48 h) were examined as in panel A and the number of pixels (i.e. the fraction of the nucleus, y-axis) at a given intensity (x-axis, in arbitrary units) tabulated for the region of the cell constituting the nucleus (n>25 cells/group). Note that the control cells have a small fraction of low intensity staining (red bar) that increases after ISO treatment. Panel D, shows the same data (intensity in same arbitrary units) as panel C, plotted as cumulative distribution of pixel intensity (intensities greater than 100 a.u. are included in the distribution but not shown as the lines become superimposed at very high intensities due to low frequency).

Fig. 5

Interpretation of STED images and potential for in situ chromatin measurements. Panel A, measured dimensions of chromatin fibers in vitro, which contain dozens of individual nucleosomes [29]. Panel B, the resolution of the STED microscope allows labeling of endogenous chromatin fibers (hashed cylinders; red dots are foci in STED images) which can then be traced to discern local structure (blue line). Panel C, when applied to the entire nucleus, this technique in principle allows for determination of in situ chromatin architecture in cardiac and other cells.