Region-specific targeting of SERCA and RyRs

We and others have previously demonstrated that within pulmonary arterial myocytes the peripheral SR proximal to the plasma membrane preferentially incorporates dense clusters of ryanodine receptor subtype 1 (RyR1) and of S/ER Ca2+ ATPase subtype 2b (SERCA2b), while RyR2s are incorporated in the extraperinuclear SR, where SERCA are few, with SERCA2a and RyR3 clusters heavily restricted to the deepest perinuclear SR17,19. Adding to this, we now identify within the same image series a distinct subtype of SERCA pump, SERCA1 (Fig. 1a, c), which together with a discrete subset of RyR1s (Fig. 1b, c) appear to line tubular networks within the boundary of the nucleus (Fig. 1a, b; Supplementary Fig. 1). This suggested that SERCA1s and RyR1s might line invaginations of the NE, the lumen of which is continuous with the SR.

Fig. 1 Discrete clusters of SERCA1 and RyR1 are targeted to the nuclear envelope. a Upper panels, left to right, bright field image of an arterial myocyte and 3D deconvolved fluorescence images (Deltavision, doconvolution) of SERCA1 labelling (green). Lower panels, left to right, digital skin encapsulating SERCA1 labelling ± nuclear labelling (blue, DAPI). b As for (a) but for RyR1 labelling (red). c Dot plot shows density of labelling (μm3 per μm3, mean ± SEM) for (upper panels) SERCA1 (n = 10 cells from 3 rats), SERCA2a (n = 12 cells from 3 rats) and SERCA2b (n = 10 cells from 3 rats) and (lower panels) RyR1 (n = 15 cells from 3 rats), RyR2 (n = 12 cells from 3 rats) and RyR3 (n = 10 cells from 3 rats) within the 4 designated regions of the cell; one-way ANOVA followed by a Tukey post-hoc test: *p < 0.05, **p < 0.01, ***p < 0.001 Full size image

SR junctions demarcate networks of cytoplasmic nanocourses

We explored this possibility using Fluo-4 to report on Ca2+ signals and a membrane permeant DNA marker (Draq5) to identify the boundary of the nucleus. By adjusting the threshold for fluorescence detection, it was evident that a network of narrow tributaries of cytoplasm ≤500 nm wide penetrated the nucleus, perhaps reflecting the path of nuclear invaginations. The cytoplasmic nanocourses within the boundary of the Draq5 labelled nucleus exhibited markedly higher levels of Fluo-4 fluorescence than the surrounding nucleoplasm, and could be equally well distinguished from any aspect of the wider cytoplasm, which in turn and invariably exhibited higher basal fluorescence than the nucleoplasm (Fig. 2a). This suggested that invaginations of the NE might demarcate discrete signalling compartments that could be observed without the need for further image processing, irrespective of whether or not differences in fluorescence intensity resulted from differences in local cytoplasmic Ca2+ concentration or the influence of the local environment within each of these compartments on general Fluo-4 fluorescence characteristics25,26. However, with the confocal system (see Methods) set to detect these cytoplasmic nanocourses within the boundary of the nucleus, we frequently observed variegated, region-specific differences in Fluo-4 fluorescence intensity in the bulk cytoplasm (beyond the boundary of the nucleus), i.e. highly localised, time-dependent and asynchronous fluctuations in Fluo-4 fluorescence intensity were evident across the wider cell. Therefore, we carried out deconvolution of all Fluo-4 images within each time series acquired. This revealed a cell-wide network of well-defined cytoplasmic nanocourses (≤400 nm across; Fig. 2a, threshold and F max set to highlight nanocourses; Supplementary Movie 1) that appeared to be demarcated by the SR (Supplementary Fig. 2). In short, different cytoplasmic nanocourses exist proximal to the plasma membrane, within extraperinuclear and perinuclear regions of cells and also penetrate the nucleus (identified by Draq5, blue, Fig. 2a). During short time series’ (2–6 min; note, experiment duration limited by photo-toxicity) hotspots of local Ca2+ flux, ≈200–400 nm in diameter, were readily identifiable in pseudocolour representations of this cell-wide network at rest (Fig. 2b), the fluorescence signal intensity of which oscillated without propagating beyond the nanocourse within which they arose. Each individual hotspot of Ca2+ flux exhibited asynchronous temporal characteristics when compared to adjacent hotspots within the same nanocourse, or hotspots arising in different nanocourses (Fig. 2b, c; Supplementary Movies 2–4). Such activity was not evident in averages of Fluo-4 fluorescence for any given nanocourse as a whole (Fig. 2c, lower panels). Notably, distances of separation between hotspots for subplasmalemmal (359 ± 15 nm) and nuclear nanocourses (350 ± 13 nm; Fig. 2d) are consistent with those for skeletal muscle RyR1s27, while distances of separation for extraperinuclear (414 ± 22 nm) and perinuclear (452 ± 32 nm) nanocourses (Fig. 2d) are significantly greater and consistent with those for cardiomyocyte RyR2s (0.6–0.8 µm)28. This is in accordance with previous studies on the spatial organisation of RyRs in the cells studied here (Fig. 1b, c). Hotspots of fluorescence were markedly attenuated by prior depletion of SR Ca2+ stores using thapsigargin, a SERCA inhibitor (Fig. 2e and Supplementary Fig. 3), and upon blocking RyRs with tetracaine (Fig. 2e; note, following pre-incubation with tetracaine hotspots within nuclear nanocourses remained visible, but neither hotspots nor nanocourses could be reliably identified outside the boundary of the nucleus, where measures had to be taken within regions of interest under these conditions). As indicated above, nanocourse hotspots exhibited clear, time-dependent fluctuations in fluorescence intensity (Fig. 2f and Supplementary Fig. 4). Irrespective of the nanocourse in question, at least two discrete levels of hotspot intensity (ΔF H /F N0 ; H = hotspot; N 0 = nanocourse at 0 s) were evident despite the limited temporal resolution at the optimal sampling frequencies used here (0.5 Hz; scan speed limited by signal-to-noise). Transitions from basal to the highest level of fluorescence intensity (ΔF x /F 0 , mean ± SEM: peripheral 0.18 ± 0.02; extraperinuclear 0.15 ± 0.01; perinuclear 0.16 ± 0.02; nuclear 0.15 ± 0.02; n = 7 cells from 7 rats) varied in duration from ~2 s to ≥10 s, with even longer dwell times evident for lower frequency, low intensity sub-states. The asynchronous activity, spatial characteristics and pharmacology of hotspots suggests that these events most likely reflect low level, basal Ca2+ flux (leak) from the SR via RyRs. However, while RyRs can remain open for many seconds, the fastest gating events are on the millisecond time scale29. Therefore, the development of confocal systems with higher temporal and spatial resolution is required before we can measure the kinetics of hotspots of Ca2+ flux characterised here with precision and thus confirm whether they truly represent unitary Ca2+ release through RyRs. Nevertheless, it is clear that the Ca2+ signalling machinery of subplasmalemmal, extraperinuclear, perinuclear and nuclear nanocourses incorporates unique receptor components, conferring different nanocourses with the capacity to deliver discrete spatially- and functionally-segregated signals. Supporting the view that the wider network of cytoplasmic nanocourses may represent a circuit for cell-wide communication, LysoTracker Red labelled endolysosomes migrated through this network of cytoplasmic nanocourses (Fig. 2g; 0.25 Hz sampling frequency for dual labelling; Supplementary Movie 5). By contrast, in these differentiated cells MitoTracker Red labelled mitochondria formed static clusters, as reported previously by others30, that sat within the nanocourse network (Fig. 2h; Supplementary Movie 6).

Fig. 2 SR Ca2+ flux within a cell-wide circuit of cytoplasmic nanocourses of arterial myocytes. a (left to right), confocal z sections through acutely isolated arterial myocyte loaded with Fluo-4 (green, calcium indicator) and Draq 5 (blue, nucleus), then deconvolved, and pseudocolor applied to show relative Fluo-4 intensity. Regions of interest identify exemplar subplaslemmal (white), extraperinuclear (blue), perinuclear (green) and nuclear (yellow) nanocourses. b (left to right), nanocourses in (a) at higher magnification and different time points; note, thresholds set independently to visualise hotspots. Grey circles identify hotspots (H1, black; H2, orange) of Ca2+ flux in exemplar nanocourses. c Fluo-4 fluorescence ratio (F x /F 0 ; where F 0 = fluorescence at time 0 and F x = fluorescence at time = x) versus time (sampling frequency = 0.5 Hz) for H1 and H2 (upper panels, left to right) and the average for the whole nanocourse (lower panels, left to right). d Scatter plot shows distances separating hotspots (mean ± SEM; ≥36 hotspots, n = 7 cells from 7 rats) within subplasmalemmal (white), extraperinuclear (blue), perinuclear (green) and nuclear (yellow) nanocourses. e Dot plots show the effect of thapsigargin (1 µM; 30 min pre-incubation; n = 3 cells from 3 rats) and tetracaine (1 mM; 4 h pre-incubation; n = 5 cells from 4 rats) on the amplitude (mean ± SEM) of Fluo-4 fluorescence ratio change (ΔF X /F 0 ); t-test with Welch’s correction: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. f Image time series highlights (white rectangle) time-dependent intensity fluctuation of one hotspot in a different subplasmalemmal nanocourse (arrow in (a), upper panel, right most image), with a record of fluorescence intensity against time (ΔF H /F N0 ; H = hotspot, N 0 = nanocourse at time = 0) from basal (B) to high intensity (H) states; note prolonged sub-state. g Deconvolved time series of z sections (0.25 Hz) show LysoTracker Red labelled endolysosomes in cytoplasmic nanocourses identified by Fluo-4 (confirmed in 3 cells from 3 different animals). h As for (g), but for mitochondria labelled with MitoTracker Red (confirmed in 3 cells from 3 different animals). Pseudocolour look up tables in (a) and (b) indicate relative fluorescence intensity in arbitrary units Full size image

Unloading SR Ca2+ into PM-SR junctions relaxes smooth muscle

We were able to confirm site- and function-specific signalling by employing Maurocalcine, a membrane-traversing peptide from scorpion venom that selectively activates RyR131. Accordingly, Maurocalcine preferentially directed increases in Ca2+ flux into subplasmalemmal nanocourses (Fig. 3a–c, e; Supplementary Fig. 5; Supplementary Movie 7), which evoked concomitant myocyte relaxation (Fig. 3d). By contrast there was relatively little change in Ca2+ flux within even the most proximal extra/perinuclear nanocourses. We have therefore visualised for the first time unloading of SR Ca2+ through RyR1s into the ‘superficial buffer barrier’ demarcated by PM-SR junctions32, which confer nanoscale path lengths and have long been predicted to coordinate Ca2+ removal from the SR and thus relaxation, as well as SR refilling during prolonged contraction5,33,34. This relates clinically35, confirming that β-adrenoceptors promote pulmonary artery dilation through RyR1-mediated Ca2+ release into PM-SR junctions of pulmonary arterial myocytes, for onward removal across the PM through forward mode Na+/Ca2+-exchanger activity16,17. Curiously, however, over the time course of our experiments (2–6 min) Maurocalcine-induced myocyte relaxation was not accompanied by concomitant falls in Ca2+ flux into extra/perinuclear nanocourses. If anything, asynchronous Ca2+ flux continued within these nanocourses, with perhaps slight increases in activity but no evidence of cell-wide signal propagation (Fig. 3c, Supplementary Movie 7). One explanation for this could be that the relatively small population of RyR1s in extra/perinuclear nanocourses neither face nor couple with the contractile apparatus, but act instead to direct Ca2+ flux towards PM-SR junctions via SERCA2b and away from SR release sites occupied by RyR2s/RyR3s that guide myofilament contraction (see below). Consistent with the spatial separation of RyR1s (Fig. 1a–c), Maurocalcine also evoked marked increases in Ca2+ flux into nuclear nanocourses adjacent to relatively inactive perinuclear nanocourses, which, therefore, neither generated nor received these signals (Fig. 3a–c, f; Supplementary Fig. 6; Supplementary Movie 7). This exposes the functional segregation of nuclear nanocourses from their nearest neighbour, through the strategic targeting of RyR1s to the ONM that demarcates nuclear nanocourses (see below). It is also evident that Maurocalcine-evoked Ca2+ flux within nuclear nanocourses did not propagate freely into the nucleoplasm to any great extent, i.e. Ca2+ is released across the ONM into the cytoplasmic nanocourses defined by each invagination but not directly into the nucleoplasm (Fig. 3a, c, e, f). Consistent with outcomes for nanocourse Ca2+ flux at rest, all responses to Maurocalcine were blocked by tetracaine and by prior depletion of SR stores with thapsigargin (Fig. 3f).

Fig. 3 Maurocalcine gates Ca2+ flux into subplasmalemmal nanocourses and nuclear invaginations. a (upper panels) Deconvolved confocal images show pseudocolour representations of Fluo-4 fluorescence intensity in z sections through an acutely isolated arterial myocyte (white broken line identifies nucleus) before and during application of 300 nM Maurocalcine (white arrow). White boxes inset show example subplasmalemmal nanocourses at higher magnification. b From left to right, high magnification examples of subplasmalemmal (white), extraperinuclear (blue), perinuclear (green) and nuclear (yellow) nanocourses identified by regions of interest in (a), at three different time points. Grey circles identify for each nanocourse, two hotspots (H1, black; H2, orange) of Ca2+ flux. c Fluo-4 fluorescence ratio (F x /F 0 ; where F 0 = fluorescence at time 0 and F x = fluorescence at time = x) versus time (sampling frequency = 0.5 Hz) for H1 and H2 of each nanocourse (upper panels, from left to right) compared to the average for the whole nanocourse (lower panels, from left to right). d Dot plot shows cell area (µm2; mean ± SEM) before and after extracellular application of 300 nM Maurocalcine (n = 3 cells from 3 rats). e Dot plot shows peak change (ΔF x /F 0 ; mean ± SEM; n = 3 cells from 3 rats) for Fluo-4 intensity for hotspots and nanocourses within each region of interest at the peak of the response to Maurocalcine (300 nM). f As for (e) but for whole nanocourses in the absence and presence of thapsigargin (1 µM, 30 min pre-incubation; n = 4 cells from 3 rats) or tetracaine (1 mM; 4 h pre-incubation; n = 4 cells from 4 rats); t-test with Welch’s correction: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The pseudocolour look up tables in (a) and (b) indicate relative fluorescence intensity in arbitrary units Full size image

Nuclear invaginations delimit diverse nanocourse networks

Using electron microscopy we observed 20–200 nm diameter invaginations, as have others36, within the nucleus of arterial myocytes in-situ in arterial sections. We could distinguish invaginations of the ONM, forming open transnuclear channels, or shallow, blind invaginations of variable depths reaching into the nucleus (Fig. 4a). As the NE is a double membrane, invaginations also contained inner nuclear membranes (INM) with the luminal space between INM and ONM ranging from 10 to 50 nm. Staining of fixed cells for lamin A, which generally lines the INM, revealed a tubular network formed by nuclear invaginations that criss-crossed the nucleoplasm of these differentiated cells (Fig. 4b), as do nuclear nanocourses. We confirmed that this nucleoplasmic reticulum (NR) held a Ca2+ store that was continuous with the perinuclear SR by loading the S/NR lumen using a Ca2+ indicator (Calcium Orange) in the absence (Fig. 4c) and presence of SR staining (ER Tracker; Fig. 4d). Therefore, pulmonary arterial myocytes specifically target SERCA1 and RyR1 to the NE in order to facilitate signal segregation within those cytoplasmic nanocourses demarcated by invaginations of the NE.

Fig. 4 Nuclear invaginations demarcate a releasable Ca2+ store and cytoplasmic nanotubes. a Electron micrographs of artery sections, show (left to right) arterial smooth muscle cells at low and high magnification and identify invaginations (I) of the inner (INM) and outer (ONM) nuclear membrane: PM plasma membrane, C cytoplasm, M mitochondria, N nucleus; confirmed in 4 arteries from 4 rats. b Left hand panel shows 3D reconstruction of a deconvolved z stack of confocal images through the nucleus of an arterial myocyte labelled for lamin A (red) with (left panel) and without (middle panel) DAPI (blue) to identify the nucleus (N) and its invaginations (I); confirmed in 54 cells from 14 rats. Right panel, higher threshold and ‘digital surface skin’ applied to select for nuclear invaginations by way of their higher density of labelling for lamin A. Then, higher magnification transverse section through the 3D image of lamin A labelling shown at 2 different angles. c (left to right), 3D reconstruction of a deconvolved z stack of confocal images showing Calcium Orange fluorescence (orange) from within the lumen of the sarcoplasmic (SR) and nucleoplasmic reticulum (SR) of an arterial myocyte, with the nucleoplasm identified (Draq5, blue), higher magnification transverse section through the nucleus of same cell without Draq5 (N, nucleus; I, invaginations), application of digital skin (30° image rotation) and longitudinal section through the centre of the nucleus, then a transverse section through the nucleus (45° image rotation); confirmed in 5 cells from 3 rats. d (from left to right), Deconvolved confocal z section through the middle of a pulmonary arterial myocyte showing ER-tracker identified SR and outer nuclear membrane (white), Calcium Orange fluorescence (orange), merged image showing ER-tracker and Calcium Orange fluorescence, higher magnification images with Draq5 identifying the nucleus and its invaginations (N, nucleus; I, invaginations), and a 90° rotation; confirmed in 4 cells from 3 rats Full size image

Closer inspection of Ca2+ flux within nuclear nanocourses revealed functional signal segregation in response to not only Maurocalcine (Fig. 3; Supplementary Fig. 6; Supplementary Movie 8) but also to the vasoconstrictor Angiotensin II (Fig. 5a, b; Supplementary Movie 9). Both stimuli triggered increases in Ca2+ flux within a subset of nuclear nanocourses, and with distinct spatiotemporal signatures evident in each of these activated nanocourses (Fig. 5b; Supplementary Fig. 6; Supplementary Movies 8 and 9).

Fig. 5 Ca2+ flux into nuclear invaginations regulates gene expression. a (from left to right) Confocal z section of Fluo-4 fluorescence in an arterial myocyte (green) ± nuclear label (blue, Draq5), indicating perinuclear cytoplasm (C), nuclear invaginations (I1, I2, I3, I4) and nucleoplasm (N), and fluorescence intensity plot along vertical dashed black line marked in images. b Time series of 3D intensity maps for nuclear region of cell in (a) during application of Angiotensin II (30 μM, white arrow). c (from left to right) 3D reconstruction of section through the nucleus of a myocyte labelled for lamin A (red; confirmed in 54 cells from 14 rats) and showing co-localisation with H3K9me2 (white; confirmed in 14 cells from 5 rats), then same image with digital skin, sectioned and rotated to identify a transnuclear invagination. d, e As in (c) but different cells. f–h As in (c–e), but showing BAF co-localisation with emerin; confirmed in 10 cells from 4 rats. i, j Dot plots show (mean ± SEM) the effect of blocking RyRs with tetracaine (TTC, 1 mM, 90 min pre-incubation) on MLH1 and S100A9 expression in acutely isolated pulmonary arterial myocytes, assessed by i q-RT-PCR (assayed in triplicate, for n = 3 rats) and j RNAscope (counts per cell, 14–57 cells per plate, n = 9 independent experiments from 3 rats); t-test with Welch’s correction: **p < 0.01. The green and pseudocolour look up tables in (a) and (b) indicate relative fluorescence intensity in arbitrary units Full size image

The functional reasons for the isolation of nuclear nanocourses are not clear, but it may be to prevent wide-scale gene activation/inactivation events that could switch cells from a differentiated to proliferative phenotype, operated through specific changes in Ca2+ flux. Normal ovoid nuclei tend to have histones carrying both H3K9me2 and H3K9me3 marks, and the chromatin cross-linking protein barrier to auto-integration factor (BAF) associating with NE proteins such as emerin and making the nuclear periphery generally silencing37,38,39. However, interestingly, these marks segregate in differentiated arterial myocytes with the H3K9me2/3 both still at the outer limits of the nucleus but depleted with respect to BAF, and the nuclear invaginations rich with H3K9me2 (Fig. 5c–e) and BAF (Fig. 5f–h) but depleted with respect to H3K9me3 (Supplementary Fig. 7). The combination of H3K9me2/3 together is strongly silencing, but absent the me3 mark and the me2 can reflect a poised state that has been found at myogenic regulators such as the myogenin promoter40. It is possible that the non-propagating Ca2+ transients in distinct invaginations in some way specifically regulate chromatin in differentiated cells as the different chromatin marks are concentrated in puncta (Fig. 5c–h): discrete H3K9me2-lamin A puncta (471 ± 38 nm in diameter) were separated by 335 ± 46 nm, while emerin-BAF puncta (361 ± 41 nm in diameter) were separated by 495 ± 61 nm, approximating the 350 nm spacing between the tetracaine-sensitive hotspots of nuclear nanocourses. Potentially Ca2+ and/or charge responsive and functionally distinct chromatin domains may therefore be established by nuclear invaginations. Supporting this, qRT-PCR (Fig. 5i) and RNAscope® (Fig. 5j) showed that blocking Ca2+ flux through RyRs with tetracaine (1 mM, 90 min pre-incubation) reduced the expression of two genes of interest (identified by RNAseq), one encoding the DNA mismatch repair protein MutL homolog 1 (Mlh1), which can be repressed through interaction with H3K9me241,42, and another encoding the S100 calcium binding protein A9 (S100a9), which can be repressed by BAF43. That said, further investigation into the role of nuclear invaginations in regulating gene expression will undoubtedly reveal greater complexities of gene regulation, given that individual, acutely isolated smooth muscles possess different types (Supplementary Fig. 8) and different numbers of lamin A and emerin positive invaginations.

Cell-wide signal propagation and smooth muscle contraction

As one might expect, increases in Ca2+ flux within nuclear invaginations induced by Angiotensin II were accompanied by a Ca2+ wave that propagated through all extraperinuclear and perinuclear nanocourses, triggering concomitant myocyte contraction that was evident from reductions in cell surface area (Fig. 6a–c; Supplementary Movie 10; note, when the threshold and F max are set to limit signal saturation, nanocourses are not so clear at rest, see Supplementary Fig. 9). These events were immediately preceded by a rapid fall in Fluo-4 fluorescence intensity within the majority of cytoplasmic nanocourses, except for those at the point of wave initiation, suggesting that Angiotensin II also acts to pre-load the SR with Ca2+; perhaps a critical step prior to induction of cell-wide signal propagation. We were unable to study the action of Angiotensin II in the absence of myocyte contraction, because Wortmannin disrupted nanocourse arrays and ML-9 alone did not block cell contraction observed at room temperature. Nevertheless, closer inspection of the Ca2+ wave revealed that while marked increases in Fluo-4 fluorescence were recorded in extra/perinuclear regions (Fig. 6a, b, e), there was little or no increase in Ca2+ flux into subplasmalemmal nanocourses demarcated by PM-SR junctions (Fig. 6a (upper panels), b, e), which are key to myocyte relaxation (see Fig. 3). This is significant given that RyR2 and RyR3 are preferentially targeted to extraperinuclear and perinuclear regions, respectively, while RyR1 clusters predominate in subplasmalemmal regions and nuclear invaginations17,19, because it is RyR2 and RyR3, but not RyR1, that hold the capacity to carry propagating waves by Ca2+-induced Ca2+ release (CICR)18,28,44,45. Irrespective of cellular region, increases in Ca2+ flux induced by Angiotensin II were abolished by prior depletion of SR stores with thapsigargin, block of RyRs with tetracaine and by pre-incubation with the cyclic ADP-ribose antagonist 8-bromo-cADPR (Fig. 6d, e), which is in line with the fact that 8-bromo-cADPR also blocks hypoxic pulmonary vasoconstriction at the level of the smooth muscle46,47. Accordingly, we have previously shown that intracellular dialysis of high concentrations of cADPR (100 μM) evokes a global Ca2+ wave and contraction of acutely isolated pulmonary arterial myocytes47,48. Given that cADPR preferentially activates RyR1s and RyR3s49 but can only sensitise RyR2s to CICR44, it therefore seems likely that cADPR accumulation within or proximal to extraperinuclear nanocourses in response to Angiotensin II, serves to activate local subpopulations of RyR1s and/or RyR3s49 while delivering concomitant sensitisation of RyR2s to`CICR44, that permits subsequent initiation of a propagating Ca2+ signal and thus myocyte contraction. This is in stark contrast to the effect of low concentrations of cADPR (10 μM), the intracellular dialysis of which preferentially releases Ca2+ from RyR1s on the peripheral SR proximal to the plasma membrane, to thus evoke membrane hyperpolarization and vasodilation16,17. Furthermore, the response to Angiotensin II remained unaffected in the presence of the inositol (1,4,5) trisphosphate receptor (IP 3 R) antagonist 2APB (not shown). This is in accordance with the fact that IP 3 Rs do not couple by CICR to RyRs in pulmonary arterial myocytes50,51, and suggests that this segregation of RyRs from IP 3 Rs might be conferred by the targeting of RyR2/RyR3s to the SR that demarcates cytoplasmic nanocourses.

Fig. 6 Angiotensin II induces myocyte contraction by directing propagating Ca2+ signals through nanocourses. a (upper panels), Time series of deconvolved z sections shows pseudocolour representations of Fluo-4 fluorescence intensity in an arterial myocyte (white broken line indicates nucleus) during Angiotensin II application (30 µM, white arrow). Insets above and below image show example subplasmalemmal nanocourses at higher magnification. Lower panels as in upper panels but showing changes in Fluo-4 fluorescence intensity within nuclear nanocourses (white arrows indicate: nucleoplasm, N; Nuclear nanocourses, NI1, NI2 and NI3). Note, with cell-wide acquisition of Angiotensin II responses, signal saturation (grey bar) in some nuclear nanocourses was unavoidable (excluded from quantitative analysis). b Fluo-4 fluorescence ratio (F x /F 0 ; where F 0 = fluorescene at time 0 and F x = fluorescence at time = x) versus time (sampling frequency 0.5 Hz) for a subplasmalemmal nanocourse (white), regions of interest within the extraperinuclear (blue) and perinuclear (green) areas of the cell (extra/perinuclear nanocourses could not be followed during contraction), and for nuclear nanocourses (NI1–3, yellow) and nucleoplasm (N, black). c Dot plot shows cell area (µm2; mean ± SEM) before and after Angiotensin II (30 µM; n = 8 cells from 4 rats). d Dot plot shows basal Fluo-4 intensity (F/F NO ; mean ± SEM; n = 8 cells from 4 rats) within the nucleoplasm (black) and nuclear nanocourses (yellow). e Dot plots show peak change (ΔF x /F NO ; mean ± SEM) for Fluo-4 intensity within specified region of interest after Angiotensin II (30 µM; n = 8 cells from 4 rats), in the absence and presence of thapsigargin (1 µM; 30 min pre-incubation; n = 5 cells from 5 rats), tetracaine (1 mM; 4 h pre-incubation; n = 4 cells from 4 rats) and 8-bromo-cADPR (100 µM; 30 min pre-incubation; n = 3 cells from 3 rats); t-test with Welch’s correction: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The pseudocolour look up table in (a) indicates relative fluorescence intensity in arbitrary units Full size image

That the widths of all extra/perinuclear nanocourses are on the nanoscale (≈500 nm across) is consistent with the finding that the functional Ca2+-binding protein calmodulin is tethered proximal to the SR membranes that line myofilament arrays52, rather than being freely diffusible in the cytoplasm. All relevant path lengths from the SR to myofilaments must therefore be on the nanoscale too. Multiple coordinated actions may thus be delivered by signal segregation between distinct nanocourse networks, enabling nanocourse-specific delivery of Ca2+ signals with distinct temporal characteristics (Figs. 3, 5 and 6, Supplementary Fig. 10).

Remodelling of nanocourse networks upon cell proliferation

During the transition to proliferating myocytes in culture, the entire, cell-wide network of cytoplasmic nanocourses was dismantled, inclusive of the rapid loss (≤24 h) of lamin/emerin positive nuclear invaginations (Fig. 7a–i; Supplementary Fig. 11); although in some cells one single lamin/emerin negative, transnuclear invagination was identified by ER tracker staining, although these do not appear to support Ca2+ signalling (Supplementary Fig. 12). That this phenotypic change is delivered through reconfiguration of the cell-wide nanocourse network that directs Ca2+ flux is further highlighted by: (i) A switch in dependency of Angiotensin II-induced Ca2+ transients from RyRs to IP 3 Rs (Fig. 7j–l); (ii) Unrestricted, cell-wide SR Ca2+ release due to loss of cytoplasmic nanocourses; (iii) Loss of the ‘nuclear buffer barrier’53 that opposed direct Ca2+ flux into the nucleoplasm in acutely isolated cells (Fig. 7j–l). Accordingly, others have found that myocyte proliferation coincides with whole-scale changes in gene expression inclusive of a decrease in lamin A54 and RyR expression, and augmented IP 3 R expression55. Our observations are therefore consistent with the idea that invaginations act to regulate anti-proliferative genes, that is until the proliferative phenotype56 is ready to be engaged. Unfortunately, rapid loss of nuclear invaginations prevents normal genome manipulations in cultured cells to directly test this proposal. Nevertheless, qRT-PCR showed that during cell proliferation loss of S100A9 expression, but not MLH1 expression, was associated with loss of nuclear invaginations (Fig. 7m, n), consistent with the impact on S100A9 expression of reduced Ca2+ flux through RyRs (Fig. 5i, j) and previous reports on S100A9 repression during proliferation of airway smooth muscles57. These observations, the distribution of chromatin marks and general tendency of NE-association to keep chromatin repressed58,59 lends support to the view that NE invaginations may play a role in genome regulation and cycles of gene repression and activation.