Nuclear connectin novex-3 promotes proliferation of hypoxic foetal cardiomyocytes

Figures

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  Figure 1. Novex-3 is localised in the nucleus of mouse fCMs. (a) A representative immunoblot for novex-3 protein (marked with *) from foetal mouse heart tissues (left and right) or cultured mouse fCMs (middle, E17). The subcellular fractionation of proteins was done by NE-PER system, which uses CER buffer for cytoplasmic extraction and NER buffer for nuclear extraction (left and middle). In some samples, CER buffer was replaced with MPER buffer to validate the result of different fractionation methods (right; see Methods). The broken line between the lanes separates the samples from different parts of the same blot. (b) Detailed fractionation analysis was performed to obtain five fractions, i.e. cytoplasmic, membrane, soluble nuclear (sNuc), insoluble chromatin-bound nuclear (iNuc), and cytoskeletal fractions, which were used to probe novex-3 protein (marked with *) by immunoblotting. (c) The two fractionation methods used in (a) were validated by immunoblotting a specific protein with known localisation: β-tubulin (cytoplasmic), nup98 (nuclear) and fibrillarin (nuclear). (d) The fractionation method used in (b) was validated by immunoblotting a specific protein with known localisation:…

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  Figure 2. Novex-3 nuclear localisation appears to be regulated by the nuclear localisation signal (NLS) in the N-terminal region. (a) Primary structure of human novex-3 (assembled from refs20,36). Mouse novex-3 has a similar molecular structure (Supplementary Fig. S9). The epitope of the two different antibodies used in this study is marked with “e1” and “e2”, both of which is within the novex-3-specific exon (see Methods). The nuclear localisation signal (NLS) located at N-terminus between Z2 and Z repeats17 is shown. (b) Four fragments covering the full length novex-3 (novex-3-1 through novex-3–4) as well as novex-3-Nterm fragment containing connectin exons 1 to 6 (shown in (a)), were independently expressed as N-terminal EGFP-tagged proteins in cultured fCMs (E16–E18) by baculovirus-mediated protein expression system. Live cells were observed by fluorescence microscopy together with bright-field optics. The approximate protein size of these five fragments, excluding the tagged-EGFP (~27 kDa), was 146, 206, 170, 146 and 32.6 kDa for novex-3–1, novex-3-2, novex-3-3, novex-3–4 and novex-3Nterm, respectively. (c) The E16 fCMs doubly expressing novex-3-Nterm-GFP and sarcomeric…

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  Figure 3. Nuclear expression of novex-3 in hypoxic fCMs is repressed after birth. (a) Immunofluorescence staining for novex-3 (green) in phalloidin (red) and DAPI (blue)-stained mouse heart sections at indicated stages. E; embryonic day, P; postnatal day. Scale bar = 20 µm. (b) qPCR analysis of novex-3 mRNA expression in mouse hearts at indicated stages. n = 4–6 independent experiments. ***P < 0.001 compared to E17. Error bar = SEM. (c) A representative immunoblot for novex-3 protein in nuclear fraction from mouse hearts at indicated stages. Histone H3 was included as a loading control.

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  Figure 4. Nuclear expression of novex-3 in hypoxic fCMs is repressed by exposure to higher O2. (a) Immunofluorescence staining for novex-3 (green) observed in phalloidin (red) and DAPI (blue) in E17 fCMs cultured under different O2 conditions. Scale bar = 20 µm. (b) Immunofluorescence staining for novex-3 (green, another e2 antibody was used; see Methods) observed in sarcomeric α-actinin (red, as a CM marker) and DAPI (blue) in E17 fCMs cultured under different O2 conditions. Scale bar = 20 µm. (c) qPCR analysis of novex-3 mRNA expression in fCMs cultured under different O2 conditions. n = 3 independent experiments. **P < 0.01 compared to 3% O2. Error bar = SEM. (d) Immunoblot analysis for novex-3 protein in nuclear fraction from E17 fCMs cultured under different O2 conditions. Histone H3 was included as a loading control.

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  Figure 5. Nuclear novex-3 is a cell cycle promoter in hypoxic fCMs. (a) Novex-3 was silenced in E17 fCMs by siRNA, and the nuclear fraction was used to probe novex-3 protein by immunoblotting. Two siRNAs specific for novex-3, designated as si-1 and si-2, were used. Histone H3 was included as a loading control. (b) qPCR analysis of mRNA levels of the indicated genes in novex-3-silenced fCMs (E16–E17). Data are shown as normalised to the si-control level of each gene, set at 1. n = 3 independent experiments. ***P < 0.001 compared to the si-control level of each gene. (c,d) Immunofluorescence for Ki67 (c, green) and phospho-histone H3 (pH3) (d), green) observed in sarcomeric α-actinin (red, as a CM marker) and DAPI (blue) in novex-3-silenced fCMs (E17). Arrows denote Ki67-positive (c) and pH3-positive (d) fCMs. Proportions of Ki67-positive (c) and pH3-positive (d) fCMs were shown on the right. n = 4 independent experiments. *P < 0.05 compared to si-control. Scale bars = 50 µm. (e) Proliferative activity of novex-3-silenced E16–E17 fCMs evaluated by cell counting after 72–96 h culture. n = 3 independent experiments. **P < 0.01 compared to si-control. (f,g) Immunofluorescence for Ki67 (f),…

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  Figure 6. Nuclear function of novex-3 as a cell cycle promoter is ascribed to that in interphase, but not during mitosis. (a) A representative time-lapse recording of cell division dynamics of the E16 fCM doubly expressing novex-3-Nterm-GFP and sarcomeric α-actinin-mCherry (as a CM marker). In contrast to the clear nuclear expression observed during interphase (arrow), novex-3-Nterm expression was not localised to specific structures (spindles, spindle matrix, centrioles, or chromosomes) during mitosis; instead, it was diffusely spread throughout the cytoplasm following nuclear envelope breakdown at prometaphase. At telophase, when the nuclear envelope reassembled, the novex-3-Nterm re-accumulated in the newly formed daughter nuclei (arrows in telophase and cytokinesis), where it was maintained over the next interphase (arrows). The number on each panel indicates the time in (hours: minutes) elapsed from the time indicated in the first panel. Scale bar = 50 µm. (b) Cultured fCMs (E17–E18) were triple-stained with novex-3, sarcomeric α-actinin (as a CM marker), and DAPI. DAPI staining clearly defined each phase in mitosis, including prophase, metaphase, anaphase, and subsequent cytokinesis.…

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  Figure 7. Novex-3 plays a role in providing the elasticity/compliance to the fCM nuclei. (a) Mechanical indentation measurements by atomic force microscopy (AFM). Intrusion of the AFM cantilever onto the surface of the fCM generated a force-indentation curve, which is the relationship between the cantilever deflection and its indentation depth. The elastic modulus (E) was calculated based on the Hertz model (see Methods). Measurements were performed on the central region of the nuclei, as monitored by phase contract optics as previously described26–28. (b) The elastic modulus of novex-3-silenced E16 fCMs evaluated on the central region of the nuclei. fCMs were cultured under 3% O2 condition. n = 6–8 fCMs. **P < 0.01 compared to si-control. Error bars = SEM. (c) The elastic modulus of E17 fCMs cultured under different O2 conditions evaluated on the central region of the nuclei. n = 6–7 fCMs. *P < 0.05 compared to 3% O2 condition. Error bars = SEM. (d) Microneedle-based tensile tests. One microneedle, designated as an operation microneedle, was rigid and was moved with a three-axis motorised micromanipulator to stretch the nucleus. The other microneedle, designated as a deflection…

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  Figure 8. Novex-3 knockdown results in reduced phosphorylation and enhanced multimerisation of nuclear lamins in fCMs. (a) Immunoblot analysis for phospho-lamin A/C at Ser-392 in nuclear fraction from novex-3-silenced E17 fCMs. Histone H3 was included as a loading control. Quantitative analysis was shown on the right. n = 5 independent experiments. *P < 0.05 compared to si-control. Error bar = SEM. (b) Immunofluorescence staining for phospho-lamin A/C at Ser-392 (green) observed in sarcomeric α-actinin (red, as a CM marker) and DAPI (blue) in novex-3-silenced E17 fCMs. Scale bar = 20 µm. (c) Immunofluorescence staining for lamin A/C observed in connectin C-terminus (red, as a CM marker) and DAPI (blue) in novex-3-silenced E17 fCMs. The epitope of this lamin A/C antibody was previously shown to be buried and thus inaccessible by lamin A/C multimerisation32,33. Note the weak and punctate signals, and thus higher level of local lamin multimerisation in novex-3-silenced fCM nuclei. Data were shown as 3D-reconstruction images generated using Volocity. The lowest panel shows a magnified image from the boxed region in the upper panel. Scale: One unit = 13.1 µm in the upper three panels, and 1.9…